The pupal specifier broad directs progressive
morphogenesis in a direct-developing insect
Deniz F. Erezyilmaz*, Lynn M. Riddiford, and James W. Truman
Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800
Edited by Kathryn Anderson, Sloan–Kettering Institute, New York, NY, and approved March 27, 2006 (received for review November 16, 2005)
life histories is the transcription factor broad (br), which specifies
pupal development. To determine the role of br in a direct-
developing (hemimetabolous) insect that lacks a pupal stage, we
cloned br from the milkweed bug, Oncopeltus fasciatus (Of’br). We
find that, unlike metamorphosing insects, in which br expression is
restricted to the larval–pupal transition, Of’br mRNA is expressed
during embryonic development and is maintained at each nymphal
molt but then disappears at the molt to the adult. Induction of a
supernumerary nymphal stage with a juvenile hormone (JH) mimic
prevented the disappearance of br mRNA. In contrast, induction of
nymphs caused a loss of br mRNA at the precocious adult molt.
Thus, JH is necessary to maintain br expression during the nymphal
stages. Injection of Of’br dsRNA into either early third- or fourth-
stage nymphs caused a repetition of stage-specific pigmentation
patterns and prevented the normal anisometric growth of the
wing pads without affecting isometric growth or molting. There-
fore, br is necessary for the mutable (heteromorphic) changes that
occur during hemimetabolous development. Our results suggest
that metamorphosis in insects arose as expression of br, which
conveys competence for change, became restricted to one postem-
bryonic instar. After this shift in br expression, the progressive
changes that occur within the nymphal series in basal insects
became compressed to the one short period of morphogenesis
seen in the larva-to-pupa transition of holometabolous insects.
evolution of metamorphosis ? heteromorphosis ? Oncopeltus ?
juvenile hormone ? allometry
through a metamorphosis. Regulation of stage-specific differ-
ences may be under either environmental or hormonal control,
but relatively little is known of the molecular switches involved
or how changes in the timing of these switches can lead to
evolutionary change (1). In insects, metamorphosis arose once
from a direct-developing ancestor ?300 million years ago (2). A
life histories is the transcription factor broad (br) (3–7). In both
moths and flies, epidermal expression of br is restricted to the
larval–pupal transition (3, 5–7), and its expression at this time is
required for activation of pupal-specific gene expression, as well
as suppression of larval- and adult-specific gene expression (3,
7). Accordingly, Drosophila null mutants never enter the pupal
stage; instead, they remain in a prolonged larval state (8). In
addition, gynander larvae mosaic for br null and br?tissue
produced mosaic larval and pupal tissue, respectively, at the
larval–pupal transition (9). Loss of br also prevents the larval–
pupal transition in the silkmoth; tissues that were transformed
with a vector driving br RNA interference were unable to
destroyed at metamorphosis (10).
The restriction of br expression at the larval–pupal transition
of holometabolous insects occurs through the action of two
hormones: the steroid 20-hydroxyecdysone (20E) and the ses-
quiterpenoid juvenile hormone (JH). Peaks of 20E trigger molts
ife history strategies are highly plastic within animal phyla;
some groups develop directly, whereas related taxa pass
between stages, whereas the presence or absence of JH deter-
mines the type of cuticle that is produced (11) and whether br is
metamorphosis and br expression (5, 7). As JH titers disappear
in the last larval stage, a small peak of 20E triggers ‘‘pupal
commitment’’ as it induces br (3–7). Although JH levels again
rise at the pupal molt, when they suppress precocious adult
development, JH does not suppress br at this stage. In fact,
topical application of JH during the adult molt, which normally
occurs in the absence of JH and br, causes the reinduction of br
and the production of a second pupal cuticle (7).
To determine the role of br in a nonholometabolous, direct-
developing insect, we have isolated br from the milkweed bug,
Oncopeltus fasciatus. Here, we show that, as in metamorphosing
insects, br is required for morphogenesis and that its expression
at each nymphal molt, and its expression is required for pro-
gressive changes in proportions and pattern from instar to instar.
Our results suggest that metamorphosis in insects arose as
expression of this factor, which conveys competence for change,
became restricted to one postembryonic instar.
Using PCR with a set of primers to the N-terminal Broad–
Tramtrack–Bric-a-brac (BTB) domain of br, we isolated a 600-bp
br fragment from cDNA of O. fasciatus (Of’br) (GenBank
accession no. DQ176004) and used RT-PCR to study its expres-
sion. Oncopeltus progresses through five nymphal instars (de-
fined as the form observed during each successive intermolt
stage or stadium) and then molts to the adult with functional
wings and genitalia. br mRNA first appears during embryogen-
esis and is present in the first two nymphal stages (data not
shown). Fig. 1 shows br mRNA levels at daily intervals through
the last three nymphal stages. br mRNA is present through the
third and fourth instars but is up-regulated during the molt when
the next nymphal cuticle is made (Fig. 1 A and B). By contrast,
no br mRNA is present during the latter part of the fifth nymphal
instar when the adult cuticle is made (Fig. 1C).
Because br expression was correlated with nymphal molts, but
not with the adult molt, we asked whether gain or loss of a
nymphal molt is accompanied by a corresponding gain or loss of
br mRNA expression. JH is present throughout the nymphal
stages of hemimetabolous insects but disappears in the final
instar to allow adult differentiation (12, 13). JH treatment of
Oncopeltus at the onset of the fifth instar prevents adult differ-
entiation, resulting in a supernumerary sixth-stage nymph (14).
In this situation, the duration of the fifth stadium was shortened,
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: br, broad; Of’br, br from Oncopeltus fasciatus; JH, juvenile hormone; BTB,
Broad–Tramtrack–Bric-a-brac; Acd’br, br from Acheta domesticus.
database (accession nos. DQ176003 and DQ176004).
*To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
May 2, 2006 ?
vol. 103 ?
no. 18 ?
and br was up-regulated on the last day of the instar (Fig. 1D),
a pattern similar to the earlier nymphal instars (compare with
Fig. 1 A and B). To determine whether br up-regulation occurs
only at nymphal molts, we used precocene to destroy the corpora
allata, the source of JH, in fourth-instar nymphs (15, 16). After
precocene treatment, fourth-stage nymphs molted to precocious
adults, and their br expression resembled that of a fifth-instar
nymph. That is, the duration of the instar expanded, and only
trace levels of br mRNA were detected during the molt to the
precocious adult (compare Fig. 1 E and C). Thus, br expression
apparently requires the presence of JH, which is normally
present only during a nymphal molt.
Because both JH and Of’br are absent during the adult molt,
we wanted to determine whether the removal of Of’br from a
nymphal molt was sufficient to redirect the molt to adult
differentiation. Consequently, we injected Of’br dsRNA into
staged nymphs to knock down Of’br expression. This technique
has been used effectively to remove gene function from
Oncopeltus nymphs (17). Of’br dsRNA injection into the fifth
instar (n ? 17) or latter half of the fourth instar (n ? 14) had
no significant effect on the subsequent molt. The former group
molted into normal adults, whereas the latter molted to normal
fifth-stage nymphs and then to adults. However, when Of’br
dsRNA was injected during the first half of the fourth instar,
36 of 39 nymphs molted to a fifth instar of normal size but
retained the pigmentation pattern characteristic of the fourth-
instar nymph (the 4? nymph; Fig. 2C). In addition, their wing
pads were significantly smaller than those of control fifth-
instar nymphs (compare bracket length in Fig. 2 B and C) and
had the proportions of the fourth-instar wing pad (compare
Fig. 2 C and A). To see whether the repeat of instar-specific
characteristics was unique to the penultimate (fourth)
nymphal stage, we also injected third-instar nymphs with
dsRNA within the first 24 h after ecdysis. Eighteen of 34
nymphs molted to larger versions of the third instar (3? nymph;
Fig. 2E). The wing pads of these 3? nymphs were smaller than
those of control fourths, although the overall body size of the
two groups was similar. The remaining nymphs that molted to
normal fourth instars subsequently formed 4? nymphs during
their next molt. In contrast, all of the day-3 third-instar nymphs
given Of’br dsRNA molted to normal fourth instars and then
to 4? nymphs (n ? 11). As a negative control, we injected
dsRNA made from the cricket Acheta domesticus br gene
(Acd’br) into either third-instar (n ? 12) or fourth-instar (n ?
32) nymphs of various ages. All of these animals molted to
normal fourth- or fifth-instar nymphs, respectively.
We used real-time PCR to show that Of’br dsRNA knocked
down Of’br transcript levels. In control nymphs, br transcripts on
day 4 of the fourth instar measured 4.1- and 0.9-fg relative
that were fated to form 4? nymphs because of Of’br dsRNA
treatment in the late third instar, br transcripts were undetect-
able on either day 4 (n ? 2) or day 5 (n ? 1).
These Of’br dsRNA knock-down experiments suggest that
Of’br is required for the changes in cuticle character, or heter-
omorphoses, that occur between nymphal stages. If this expla-
nation is correct, then loss of Of’br in consecutive instars should
result in further repetition of the initial nymphal pattern. To test
this prediction, we generated 3? nymphs by Of’br dsRNA injec-
tion at the onset of the third instar and then gave a second dose
are typical of two to three determinations for each instar. (D) br mRNA in
fifth-instar nymphs after treatment with 2 ?g of the JH mimic pyriproxifen
ecdysed to supernumerary sixth-instar nymphs after 4 days. (E) br mRNA
instar to cause precocious metamorphosis at the end of the fourth stadium.
if any, br was detected at the end of the instar (typical of three determina-
tions). Each cDNA sample also was separately tested for Of18S ribosomal RNA
RT-PCR analysis of Of’br mRNA during nymphal life. (A–C) br expres-
squares containing swirls of orange, unmelanized cuticle (arrow). (Inset) The
shape outlined on the dorsal thorax resembles a handbell. (B) Normal fifth-
instar nymph. The anterior two-thirds of the prothorax is entirely orange,
whereas the posterior one-third is melanized. The wing pads (in brackets) are
longer than in the fourth instar. (C) Effects of Of’br dsRNA given during the
first half of the fourth instar on characteristics of the next stage nymph (a 4?
nymph). The prothoracic melanin in the fifth stadium consisted of two black
squares with orange swirls (arrow). In addition, the wing pads were signifi-
cantly smaller (brackets) than those of a normal fifth instar. The fourth-instar
abdominal melanin pattern also was repeated in this 4? nymph (data not
shown). (D) Normal third-instar nymph. The two prothoracic tergal squares
either were completely melanized (65%; n ? 29) or had small spots of orange
shape outlined in melanin on the dorsal thorax, which resembles the profile
squares were filled or nearly filled, and the shape on the dorsal thorax more
closely resembled the outline of a candlestick. (F) Effect of two doses of
The resultant 3? nymph was the size of the fifth instar but retained the
third-instar-type prothoracic patterns and wing pad morphology. (Scale bars,
br dsRNA prevents changes in nymphal thoracic pigmentation. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0509983103Erezyilmaz et al.
of Of’br dsRNA at the onset of the 3? instar. Five of eight 3?
nymphs that were injected at the onset of the 3? instar molted to
a fifth stadium but still retained the pigmentation pattern
characteristic of a third instar (Fig. 2F). In addition, the wing
pads of these 3? nymphs were extremely reduced (Fig. 2F)
compared with the normal fifth-instar pads or those of the 4?
nymphs (Fig. 2F).
Although the premature knock-down of Of’br during nymphal
life blocked the morphological transition from one nymphal
instar to the next, it did not alter the number of nymphal instars
or the transition to the adult. For example, nymphs that went
through a progression series such as 3–4-4? or 3–3?–4 (instead of
3–4-5) subsequently underwent an adult molt. Many of these
individuals (46 of 59) died without completely shedding the last
nymphal cuticle. Of the animals that were able to emerge (n ?
13), all had typical adult pigmentation and body morphology
(compare Fig. 3 B and C with A), except that their wings were
significantly reduced and often blistered and folded. Those
receiving injections of Of’br dsRNA in the third instar were more
F compared with D). Although the disappearance of Of’br is
associated with the nymphal-to-adult molt, these data suggest
that the removal of Of’br alone is not sufficient to cause the
formation of the adult. Rather, the function of br in these more
basal insects appears to reside in orchestrating transitions within
the nymphal series itself.
In contrast to holometabolous insects, where differential
growth during postembryonic life is typically restricted to meta-
morphosis (18), differential growth in hemimetabolous insects
occurs progressively through the nymphal stages and is largely
restricted to the wing pads. When we followed the growth of the
legs and antennae after injection of Of’br dsRNA into early
fourth-instar nymphs, we found that these appendages, which
normally grow at a constant rate (1.5-fold in each molt), were
unaffected by Of’br knock-down (Fig. 4 A and B Insets). In
contrast to the legs and antennae, the wing pads grow during the
fourth instar by a factor of 2.0 and 3.0 at the base and diagonal,
respectively (Fig. 4 C and D Insets). Knock-down of Of’br at the
the respective growth ratios were 1.5 and 2.0. Therefore, Of’br
has no effect on the isometric growth of the legs and antennae
but is critical for the anisometric growth of the wings. In its
absence, wing growth becomes more isometric, like the remain-
der of the body. We were able to detect Of’br expression in all
nymphal tissues (Fig. 4E).
These studies show that in the direct-developing insect Onco-
peltus, br is expressed throughout nymphal life with high levels
the heteromorphic transitions that normally occur during
nymphal molts without interfering with the molt itself. Thus, an
ancestral role of br is to confer mutability that provides for
differential growth between nymphal instars.
After knock-down of br expression in the nymph, wing pad
growth continued but became more isometric, and its propor-
tions from the previous stage were repeated. This aspect of br
function appears to be retained during metamorphosis of the fly,
because the wings of a br allele (lacking the Z2 isoform of br) are
defective in their morphology and are shortened and ‘‘broad’’
(19–21), a phenotype similar to that seen in the wings of
Oncopeltus that lack Of’br during the third or fourth nymphal
stages (Fig. 3 E and F). In contrast to the progressive role that
Of’br plays through successive nymphal molts, the function of br
in the Drosophila wing disk is restricted to the final larval instar
as the wing disk translates patterning information to produce the
pupal wing (22). Therefore, the ancestral function of br, to
support progressive anisometric growth of the developing wing
pad over a number of instars, has been restricted to the premeta-
posterior end of the abdomen. (B) A typical adult formed from a 4? nymph produced by injection of Of’br dsRNA into an early fourth-instar nymph. The wings
were significantly reduced and were often held out to the side. Eighty-three percent (n ? 47) of the 4? nymphs died during the next molt as pharate adults. Of
underwent molts to a 3? and then a 4? nymph. Adult pigmentation and morphology was normal, but the wings were smaller and held out to a greater extent
than those of adults produced from a normal fifth-instar or 4?-instar nymph. (D–F) Typical wings of a normal adult (D), an adult that followed a 4–4? nymphal
series (E), and an adult that followed a 3–3?–4 nymphal series (F).
Wings of normal adults and those given br RNA interference as nymphs. (A) A normal, uninjected adult Oncopeltus has wings that project past the
Erezyilmaz et al.
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no. 18 ?
morphic period in the last larval instar of holometabolous
The homology of the pupal stage to a developmental stage
of hemimetabolous insects has been a recurring issue among
naturalists (23). During the latter half of the 20th century, the
prevailing theory considered the pupal state to be derived from
the final nymphal stage (24). An older idea considered pupal
development to be more akin to the events that occur during
embryonic development of direct developers (25, 26), which
has recently been expanded into the pronymph hypothesis
(27). Our data support this older idea. In both crickets and
milkweed bugs, br mRNA is present during the latter half of
embryonic development (28). In these hemimetabolous em-
bryos, this period is characterized by differential growth as the
embryo progresses from the phylotypic germ band stage to a
miniature version of the adult (27, 29). In contrast, br mRNA
is not present in the epidermis of holometabolous embryos
(30), and the growth during the corresponding phase of
embryogenesis is more isometric (31). This isometric growth
then persists through postembryonic development until br
reappears at the larval–pupal transition to help direct the
differential growth needed to generate the adult form. Be-
cause br is required for postembryonic differential growth in
the hemimetabolous insect Oncopeltus, we suggest that meta-
morphosis emerged in insects as br expression and its regula-
tion of differential growth became transposed from late em-
bryonic development to the penultimate postembryonic molt
br may confer mutability to insect life history stages through
its BTB?POZ domain, a motif implicated in the establishment
and maintenance of complex differentiated states (32). Many
BTB-containing proteins regulate complex states through
chromatin deacetylation, thereby affecting the access of sub-
sequent transcription factors to response elements (32–34). In
the context of nymphal changes in Oncopeltus, the loss of Br
may prevent the access of transcription factors to response
elements that are needed for change from one stage to the
Materials and Methods
Cloning. The br gene encodes a complex locus in which a
BTB-containing ‘‘core’’ domain is fused to one of four C2H2zinc
Acd’br. A segment of Acd’br was cloned by PCR from genomic
from embryos at the onset of katatrepsis, or dorsal closure, was
used to determine the 5? and 3? ends of the cDNA, respectively,
by using the SMART RACE cDNA Amplification Kit (Clon-
protein (GenBank accession no. DQ176003). The 55-aa zinc
finger region contains two C2H2zinc fingers that show highest
identity to the Z1 fingers found in Anopheles and Aedes Broad
with that of Aedes (94%) and Anopheles (92%).
Of’br. A segment of Of’br was also cloned by PCR from genomic
DNA by using the primers described for Acheta. The SMART
RACE cDNA Amplification Kit was again used to acquire the
5? and 3? ends of the transcript from mRNA isolated at
midembryogenesis. The predicted 118-aa Oncopeltus BTB
domain showed high identity with that of other insects: 90%
with Drosophila melanogaster and the lepidopteran species,
Bombyx mori and Manduca sexta, and 92% with the cricket A.
RNA Isolation. Individuals were staged from ecdysis, and two
individuals were homogenized at 24-h intervals thereafter. After
an initial extraction with TRIzol (Invitrogen), a second phenol-
chloroform extraction was performed. This homogenate was
DNase-treated and extracted again with phenol-chloroform.
and B) Both antennae (A) and legs (B) of uninjected nymphs (filled circles)
at each molt (Insets). ‘‘Interval’’ in Insets indicates the growth that occurs
on the growth trajectory of legs or antennae. (C and D) Effect of Of’br dsRNA
error is shown where the width of the bars is greater than the data points.
Between 23 and 31 measurements were made for each Of’br dsRNA mean;
(E) Ethidium gel showing RT-PCR of Of’br mRNA in day-4 fourth-instar body,
T2 legs, antennae, and wing pads. Data are typical of two determinations.
Effects of Of’br dsRNA on growth of the antenna, leg, and wing. (A
of metamorphosis in insects. Each image is a cross section of the second
thoracic segment. Gray arrowheads denote wing pads in nymphs, and black
arrowheads indicate wing imaginal discs. The time of hatching is indicated by
throughout the nymphal stages. This expression correlates with a bout of
differential growth that occurs late in embryogenesis, as well as the progres-
sive differential growth of the wings that occurs during the nymphal stages.
br permits differential growth of imaginal structures.
Model depicting how changes in br expression follow the evolution
www.pnas.org?cgi?doi?10.1073?pnas.0509983103 Erezyilmaz et al.
RT-PCR. RNA was subjected to further DNase treatment just
before cDNA synthesis, as described in the product literature for
Promega RNase-free DNase. cDNA was made from 1 ?g of total
RNA and random hexamers with the cDNA Synthesis Kit
(Fermentas, Hanover, MD). To detect Br BTB and core expres-
sion in Oncopeltus cDNA, we used the primers Of’br12 (5?-
CACCGAAGGCAGAAATGTTG-3?) and Of’br15 (5?-AC-
for 30 s, 63°C for 30 s, and 72°C for 30 s for 33 or 34 cycles. To
normalize the reactions, we amplified each cDNA reaction with
primers designed to amplify 18S ribosomal RNA [Of18S-B
(5?-ATGGAACAGGACCTTGGTTC-3?) and Of18S-D (5?-
GTATCTGATCGCCTTCGAAC-3?)] at the same conditions as
above for Of’br12 and Of’br15, except that the temperature was
64°C and only 17 or 18 cycles were necessary. (When 33 cycles
were used to detect Of’br, 17 cycles were used to detect Of18S
mRNA. Likewise, when 34 cycles were used to detect Of’br, 18
cycles were used to detect 18S mRNA.) This measure was taken
to control for variation in Taq activity.
No genomic DNA contamination was found in sham cDNA
reactions made without reverse transcriptase when we screened
?30% of the RNA samples. Furthermore, ‘‘no-DNA’’ controls
were included with each PCR to ensure that the observed
amplification was due to cDNA. Each time point was tested
twice, from separate pools of RNA. The PCR products were run
out on 2% gels and stained with ethidium bromide. Photographs
of the gels were scanned and inverted by using PHOTOSHOP
(Adobe Systems, San Jose, CA).
dsRNA Injections. Separate RNA strands were made from plas-
mids containing the fragments of Of’br or Acd’br by using the
MEGAscript kit (Ambion, Austin, TX), and the sense and
antisense strands were annealed as described by Hughes and
Kaufman (36). Nymphal dsRNA injections were performed
with a Hamilton syringe with a 32-gauge needle, as described
by Liu and Kaufman (37) for parental dsRNA. Nymphs were
injected into the ventral abdomen until they became distended
(D. Angelini, personal communication). Unless otherwise
indicated, nymphs were injected with 4–10 ?g of dsRNA in a
volume of 2–5 ?l made from one of the following: (i) 125 bp
of the 5? UTR plus 600 bp of 5? coding region of Of’br BTB
domain and partial core sequence, (ii) the 151-bp region of the
Of’br BTB domain, (iii) 218 bp of the Of’br 5? UTR plus the
first 107 bp of the BTB domain, (iv) an equimolar mixture of
(ii) and (iii), (v) 151 bp of the Acd’br BTB domain, or (vi) 124
bp of the 5? UTR plus ?250 bp of the Acd’br BTB domain. We
could not detect any difference in activity among the different
pieces of Of’br used for RNA interference experiments, and we
then used them interchangeably.
Real-Time PCR. The primers Of’br10 (5?-CCTCTTCGTCTCCT-
GATATG-3?) and Of’br15 (5?-ACGATTAAACGACGGC-
CAAG-3?) were used in a Smart Cycler (Cepheid, Sunnyvale,
CA) with LightCycler-FastStart DNA Master SYBR Green I
(Roche Applied Science, Indianapolis) under the following
conditions: 95°C for 600 s and then 95°C for 15 s, 64°C for 6 s,
and 72°C for 6 s repeated 45 times with 3 mM Mg and a 0.5 ?M
concentration of each primer. One-twentieth of a cDNA
reaction made from 1 ?g of total RNA was used in each PCR.
18S ribosomal RNA was used to normalize the reaction with
the primers Of18S-B and Of18S-D, except that for this reac-
tion, the annealing temperature was 65°C and the Mg con-
centration was 4 mM. After normalization, amounts were
calculated by using a standard curve made from known
concentrations of Of’br plasmid DNA.
Morphometrics. Ecdysed nymphal cuticles were hardened in
70% ethanol overnight, mounted in Fluoromount (Southern
Biotechnology Associates) and photographed at ?4 magnifi-
cation on a Nikon Optiphot microscope using a digital camera
(Sony, Tokyo) and Apple video software (Macintosh). Cuticle
dimensions were measured by using NIH IMAGE software. To
measure the wing pad diagonal, the most anterior corner of the
wing pad and opposing distalmost point of the wing pad were
Drug and Hormone Treatment. Two micrograms of Precocene II
(Sigma) was applied to the tergum in 0.2 ?l of HPLC-grade
acetone (Aldrich) with a 10-?l Hamilton syringe to nymphs
within 6 h of ecdysis to the third instar. For JH treatment, 2 ?g
Chemical, Osaka) was applied in 2 ?l of HPLC-grade acetone to
nymphs within 8 h of ecdysis to the fifth instar.
We thank Prof. Thomas Kaufman (Indiana University, Bloomington)
for the Oncopeltus stock and for encouragement and for sharing methods
and materials; Hans Kelstrup for assistance with the precocene exper-
iment; and Dr. Jason Hodin for insightful comments on the manuscript.
This work was supported by National Science Foundation Grant IBN-
9904959 (to J.W.T. and L.M.R.) and National Institutes of Health Grant
GM60122 (to L.M.R.).
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