Báo cáo khoa học: Insect cytokine, growth-blocking peptide, is a primary regulator of melanin-synthesis enzymes in armyworm larval cuticle pptx

Insect cytokine, growth-blocking peptide, is a primary
regulator of melanin-synthesis enzymes in armyworm
larval cuticle
Yosuke Ninomiya
1
and Yoichi Hayakawa
2
1 Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan
2 Department of Applied Biological Science, Saga University, Japan
Animal color patterns have evolved under various
selective pressures such as predator avoidance, sexual
selection, and thermotolerance. The variety of color
patterns reflects a complex interplay between natural
selection and color phenotypes [1]. One of the most
widespread pigments in the biological world is mel-
anin, which consists of two classes: eumelanins, which
are black or brown, and phaeomelanins, which are
red, orange, or yellow [2]. In vertebrates, melanins are
derived from the catecholamine precursors 3,4-dihyd-
roxy-l-phenylalanine (Dopa) and dopamine, which are
synthesized from tyrosine by two enzymes, tyrosinase
and Dopa decarboxylase (DDC), that convert tyrosine
to Dopa and dopamine, respectively [3,4]. In insects,
although tyrosinase may play a role in melanin synthe-
sis, tyrosine hydroxylase (TH) is important in provi-
ding the dopamine precursor. Black and brown
patterns are conserved in the cuticles of a broad range
of insect species, which suggests their evolutionary
importance as adaptive traits [4–6].
Keywords
calcium ion; epidermal cell; growth-blocking

peptide; insect; uric acid
Correspondence
Y. Hayakawa, Department of Applied
Biological Science, Saga University, Honjo-1,
Saga, 840-8502, Japan
Fax ⁄ Tel: +81 952 28 8747
E-mail:
(Received 20 December 2006, revised 20
January 2007, accepted 1 February 2007)
doi:10.1111/j.1742-4658.2007.05724.x
The cuticles of most insect larvae have a variety of melanin patterns that
function in the insects’ interactions with their biotic and abiotic environ-
ments. Larvae of the armyworm Pseudaletia separata have black and white
stripes running longitudinally along the body axis. This pattern is empha-
sized after the last larval molt by an increase in the contrast between the
lines. We have previously shown that 3,4-dihydroxy-l-phenylalanine
(Dopa) decarboxylase (DDC) is activated during the molt period by prefer-
ential enhancement of its transcription in the epidermal cells beneath the
black stripes. This study demonstrated that tyrosine hydroxylase (TH)
expression is activated synchronously with DDC. Furthermore, enhance-
ment of DDC and TH transcription is due to an increase in cyotoplasmic
Ca
2+
, which is induced by the insect cytokine, growth-blocking peptide
(GBP). Enhanced gene expression for both enzymes was induced by substi-
tution of the calcium ionophore A23187, and completely blocked by
EGTA. A GBP-induced increase in cytoplasmic Ca
2+
was seen in epider-
mal cells under the black stripes but not those beneath the white stripes,

suggesting that a difference in Ca
2+
concentration in stripe cells leads to
the specific expression of DDC and TH genes. Based on the fact that epi-
dermal cells beneath the white stripes contain abundant granules composed
mainly of uric acid, which can form a complex with Ca
2+
and hence
decrease its free concentration, we discuss the possibility that uric acid, as
well as GBP, contributes to the difference in cytoplasmic Ca
2+
within the
epidermal cells.
Abbreviations
DDC, Dopa decarboxylase; Dopa, 3,4-dihydroxy-
L-phenylalanine; GBP, growth-blocking peptide; TH, tyrosine hydroxylase.
1768 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS
Larvae of the armyworm Pseudaletia separata have a
relatively simple color pattern composed largely of
black and white stripes in the dorsal cuticle that run lon-
gitudinally along the body axis [7]. It has been reported
that dopamine melanin is the predominant black mel-
anin in insect cuticles [3,8,9]. A previous study con-
firmed this by demonstrating that DDC mRNA and
protein are expressed specifically in the epidermal cells
under the black stripes but not those under the white
stripes [7]. Enhanced DDC expression increases the dop-
amine concentration in the black-stripe dorsal cuticles.
Furthermore, we have previously shown that an insect
cytokine, growth-blocking peptide (GBP), enhances

DDC expression in the integument [10,11]. We therefore
inferred that local enhancement of DDC expression by
GBP elevates the dopamine concentration in the epider-
mal cells where dopamine melanin is actively synthes-
ized to produce the black stripes in the cuticle.
In this study, we confirmed this by demonstrating
that GBP contributes to the enhancement of gene
expression for two key enzymes of melanin synthesis,
TH and DDC, and by further examining the mechan-
ism of enhancement of expression for both enzymes in
epidermal cells beneath the black stripes. We demon-
strated that gene expression for both enzymes is
enhanced by GBP-induced elevation of cytoplasmic
Ca
2+
concentrations in the epidermal cells.
Results
TH activity and gene expression
Prior studies have indicated that DCC expression in
the cuticle of armyworm larvae (P. separata)is
enhanced in the epidermal cells under the dorsal black
stripes during the last larval molt [7]. Because TH is
another key enzyme in the biosynthesis of Dopa and
dopamine, precursors of Dopa and dopamine melanin,
respectively, the predominant black melanin of the
insect cuticle [3,9], we measured integument TH activ-
ity during the last larval ecdysis. TH activity remained
very low in the ventral cuticles, which are without
black stripes, however, the activity in the dorsal cuti-
cles increased sharply during the last ecdysis (Fig. 1).

This pattern is also seen for DDC activity during the
last ecdysis (Fig. 1, inset).
To characterize the TH expression profile in the
armyworm larvae, we cloned its cDNA using RT-PCR
and RACE. The fact that two mRNA isoforms, one
long and one short, are expressed in the epidermal and
brain cells, respectively, is consistent with that reported
in Drosophila TH (Fig. 2) [12,13]. The predicted
sequence shares the highest similarity, 95%, with that
of the butterfly Papilio xuthus. Similarities between
P. separata TH and THs reported for other animals,
including humans, are also > 70%, i.e. 79, 78, 72, 72,
and 71% with those of Drosophila melanogaster, Apis
melifera, Rattus norvegicus, Mus musculus, and Homo
sapiens, respectively.
Using anti-TH IgG and TH cDNA, western and
northern blottings were carried out. Levels of both TH
protein and mRNA were clearly increased in the
integument of day 0 of the last instar larvae of the
armyworm (Fig. 3). Furthermore, immunocytochemis-
try and in situ hybridization showed that TH protein
and mRNA are expressed in the epidermal cells
directly under the black stripes (Fig. 4).
Mechanism of TH and DDC gene expression
in epidermal cells
To find sequence motifs commonly present in the
upstream regions of the TH and DDC genes, we per-
formed BLAST searches of the Bombyx mori genome
database to identify genes homologous to P. separata
L5D2

TH activity (pmol/min/µg protein)
0
2
4
6
Dorsal
Ventral
vitca C
D
D
n
( y
t
imom/lniµ
/
g
p
or
n
iet
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
L5D2

L6D0
L6D1
a*)
b*)
18
16
14
10
12
8
L6D0 L6D1
Fig. 1. TH activity in integuments of day 2 penultimate (L5D2),
day 0 last instar (L6D0), and day 1 last instar (L6D1) larvae. (Inset)
DDC activity in integuments from the same larval stages. a*, Signi-
ficantly different from TH activity of L5D2 larval dorsal integument
(P<0.005, Student’s t-test); b*, significantly different from TH
activity of L6D0 larval ventral integument (P<0.005, Student’s
t-test). Bar ¼ mean ± SD of five independent determinations.
Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle
FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1769
TH and DDC. Analysis of ~ 5 kb of the 5¢-flanking
region of both enzyme genes showed the presence of
five common cis-elements responsible for gene expres-
sion (Fig. 5). Three of these five cis-elements, AP-1,
Ets, and CREB, which are found repeatedly in the
5¢-flanking region, have been reported to be regulated
by Ca
2+
-dependent protein kinases [14–16]. Although
we did not find any evidence that these cis-elements

contribute to controlling gene expression in the insect
epidermal cells, this information prompted us to test
the possibility that the synchronous expression of both
enzyme genes is regulated by Ca
2+
concentration.
To test this possibility, isolated integuments were
incubated with the calcium ionophore A23187, and the
expression levels of TH and DDC genes were meas-
ured. Both gene expression levels were enhanced at 6 h
following incubation with A23187 in the dorsal integu-
ment with black stripes, but not in the ventral integu-
ment without black stripes (Fig. 6A). Because we have
previously shown that the insect cytokine GBP
strongly induces Ca
2+
influx into brain synaptosomes
[20], we examined whether incubation of the dorsal
integument with GBP enhanced the expression levels
of both genes. Incubation of isolated tissues with 1 nm
GBP elevated the expression levels of both genes,
whereas the addition of 1 mm EGTA prevented GBP-
dependent elevation of gene expression (Fig. 6B).
When the concentration of added Ca
2+
was higher
Met
219 376
Epidermis form of PsTH
Stop

1884531
5’UTR
3’UTR
5’UTR 3’UTR
219
Met
376 1728
Stop
Brain form of PsTH
Fig. 2. Gene structures of P. separata TH
expressed in integuments and brains. Epi-
dermis (AB274834) and brain (AB274835)
forms of P. separata TH genes. Note that
the epidermal form has an additional coding
sequence (156 bp portion shown by diago-
nal lines) near the 5¢)end.
L5D2 L6D0 L6D1 L5D2 L6D0 L6D1
116kDa
A
B
66kDa
42kDa
Commassie blue Western(4CN)
rRNA
DV
L5D2 L6D0 L6D1
DV D V
Fig. 3. Western (A) and northern (B) blots of
TH from P. separata penultimate and last
instar larval integuments. (A) Coomassie

Brilliant Blue-stained integument proteins
(left) and immunoblots of TH with anti-TH
IgG (right). (B)
32
P-labeled TH cDNA was
hybridized to northern blot of total RNAs
from dorsal (D) and ventral (V) integuments.
Regulation of melanin synthesis in insect cuticle Y. Ninomiya and Y. Hayakawa
1770 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS
than the concentration of EGTA, expression of both
enzyme genes was enhanced by GBP (Fig. 6B). Incuba-
tion of the ventral integument with GBP did not ele-
vate expression of either gene above the detectable
level (data not shown). The data indicate that GBP-
Anti PsTH IgG
Rabbit IgG
antisense probe
sense probe
A
B
Fig. 4. TH protein and mRNA expression in vertical sections of
integument. (A) Immunohistochemical staining of TH protein with
the P. separata anti-TH IgG. (B) In situ hybridization of TH mRNA
probed with antisense or sense probes of TH RNAs. Tissue sec-
tions were prepared from day 0 last instar larvae. Note that immu-
noreactivity and hybridization signals are much stronger in the
epidermal cells under the black stripe (as indicated by arrows) than
those under the white stripe.
-5.3 Bombyx mori DDC
ORF

-5.2 Bombyx mori TH
ORF
AP-1
Ets G motif CREB Grh
Fig. 5. Cis-elements in the 5¢-flanking region
of B. mori DDC and TH genes.
TH
DDC
Actin
B
in vitro
6h incubation
TH
DDC
Actin
A
0
1
1
1
M
m
1
A
SB

M
n
1
1

M
m
m
M
M
n
aC
lC
PBG
ATGE
2
PBG
M
n
1
M
n
PBG
AT
GE
20pmol BSA
Dorsal Ventral Dorsal Ventral
20pmol GBP
C
in vivo
6h after injection
TH
DDC
Actin
in vitro

6h incubation
Dorsal DorsalVentral Ventral
A23187 0µM A23187 50µM
Fig. 6. RT-PCR analysis of TH and DDC expression in the integu-
ment of day 1 last instar larvae. (A) Clear bands of TH and DDC
mRNAs expressed in the dorsal integument after coincubation with
the calcium ionofore A23187. (B) GBP-induced expression of TH
and DDC mRNAs was observed only when Ca
2+
is present in the
integument incubation medium. (C) GBP-induced expression of TH
and DDC mRNAs was observed only in the dorsal integument 6 h
after injection of GBP.
Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle
FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1771
dependent expression of the two enzyme genes in the
dorsal integument requires cytoplasmic Ca
2+
.
Specific expression of TH and DDC genes in the
integument
To produce black stripes, GBP has to activate expres-
sion of TH and DDC genes in the epidermal cells
directly under the black stripes. Therefore, we exam-
ined whether GBP-dependent enhancement of gene
expression occurs in the integument containing the
black stripes. Injection of 20 pmol of GBP enhanced
the expression of both enzymes in the dorsal integu-
ment containing the black stripes only (Fig. 6C), indi-
cating that GBP must increase Ca

2+
concentrations
specifically in the epidermal cells under the black
stripes.
We monitored the Ca
2+
concentration in the epider-
mal cells under both black and white stripes using a
laser confocal microscope. Addition of GBP to the
incubation medium containing isolated integuments
elevated the cytoplasmic Ca
2+
concentration in the
epidermal cells under the black stripes, but not in
those under the white stripes (Fig. 7A). Furthermore,
significant elevation of the cytoplasmic Ca
2+
concen-
tration was not seen in white stripe cells even with
addition of A23187, suggesting that calcium was not
present as free ions.
The data prompted us to examine how GBP main-
tains stable cytoplasmic Ca
2+
concentrations in epi-
dermal cells under the white stripes. We focused our
attention on the abundant granules composed primar-
ily of uric acid which have previously been found
only in the epidermal cells under the white stripes
A

a)
b) d) f)
c)
a)
b) d)
c)
e)
B
Fig. 7. Ca
2+
influx imaging of GBP (A) and
calcium ionophore (B) in the dorsal integu-
ment of day 1 last instar larvae. (A) a, c, e,
Laser transmission images show the center
of the dorsal integument; b, d, f, Ca
2+
indi-
cator Fluo-3 fluorescence (green) of the
same integument was overlaid with the
laser transmission image. Note that GBP
strongly induced the Ca
2+
influx only in the
black stripe integument (d), and the influx
was largely abolished by removal of extra-
cellular Ca
2+
by EGTA (f). (B) a, c, Laser
transmission images show the center of the
dorsal integument; b, d, Fluo-3 fluorescence

(green) of the same integument was over-
laid with the laser transmission image. Note
that calcium ionophore A23187 solubilized in
dimethylsulfoxide strongly induced the Ca
2+
influx only in the black stripe integument (d).
Control integument was treated with
Grace’s medium containing 0.1% dimethyl-
sulfoxide (the same concentration as d)
(a, b), B and W boxes indicate the black and
white stripe regions, respectively.
Regulation of melanin synthesis in insect cuticle Y. Ninomiya and Y. Hayakawa
1772 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS
and ventral epidermal cells (Fig. 8, insert) [7]. The
effect of uric acid on soluble calcium ion concentra-
tions was examined by coincubating CaCl
2
with uric
acid. We found that incubation with > 70 mgÆmL
)1
uric acid significantly decreased the free Ca
2+
concen-
tration (Fig. 8). The decrease in free Ca
2+
occurred
in a time-dependent manner: free Ca
2+
in the incuba-
tion medium had disappeared completely after coincu-

bation for 3 h (Fig. 8). Based on these observations,
we presume that the abundant uric acid granules con-
tributed to the change in cytoplasmic calcium from
the soluble to insoluble solid phase. However, we
need more data to substantiate this because we do
not have any direct evidence that a high level of uric
acid is also present in the cytosol of white stripe epi-
dermal cells.
Discussion
Prior studies indicated that, during the last larval ecdy-
sis, DDC is preferentially expressed only in the epider-
mal cells under the black stripes of armyworm last
instar larvae [7]. High expression of DDC produces
dopamine, which is a precursor for dopamine melanin,
the predominant black pigment of the insect cuticle;
the blackish color is thereby enhanced in the black
stripes. Although the DDC gene is not expressed in
detectable levels in epidermal cells under the white
stripes, uric acid accumulates and forms abundant
white granules. We therefore proposed that the differ-
ence in DDC activity and the presence of white uric
acid granules produce the black and white stripes seen
in the cuticles of armyworm larvae. However, the
mechanism by which epidermal cells actively express
the DDC gene directly under the black stripes remains
unknown. In this study, we focused on another key
enzyme, TH, which is involved in the dopamine syn-
thesis pathway, and characterized its gene structure
and expression pattern. TH expression in epidermal
cells is basically the same as that of DDC: TH is act-

ively expressed during larval ecdysis only in the epider-
mal cells under the black stripes. Furthermore, we
found that the 5¢-flanking regions of B. mori TH and
DDC genes contain five common cis-elements (AP-1,
Ets, CREB, G-motif and Grh), three (AP-1, Ets and
CREB) of which have been reported to be regulated
by Ca
2+
-dependent protein kinases [14–16]. The
importance of these cis-elements, especially in terms of
TH expression, was demonstrated, mutation of either
the AP-1 or CREB site abolished expression in adult
transgenic mice [17]. Furthermore, it has been reported
that, in Drosophila transgenic embryos, the Ddc pro-
moter-green fluorescent protein reporter gene with
point mutations in the single CREB and the three
AP-1-like consensus sites showed a marked reduction
in wound-induced activation compared with wild-type
reporter controls [18]. This information prompted us
to examine the relationship between cytoplasmic Ca
2+
concentration and the expression levels of both
enzymes. A calcium ionophore (A23187) elevated
expression of both enzyme genes, suggesting that Ca
2+
is a regulatory factor leading to optimal activation of
the expression of both genes.
The next logical step was to find the principal agent
causing cytoplasmic Ca
2+

elevation in the epidermal
cells. We considered GBP to be the most promising
candidate based on two previous observations: GBP
acted directly on the epidermal cells to induce expres-
sion of the DDC gene [11,19,20], and GBP enhanced
Ca
2+
influx into brain synaptosomes in a concentra-
0 1 60 180
Time (min)
Relative fluorescence intensity
2µm
hWit ts eriep
a
l
Bc epir
ts
k
2.0
1.8
1.6
1.4
1.2
1
Fig. 8. Affect of uric acid on free Ca
2+
concentration in the incuba-
tion medium. CaCl
2
(250 nM) was incubated with 70 mgÆ mL

)1
uric
acid for the indicated periods. Relative fluorescence intensity was
calculated as the difference between each fluorescent value and
that of the deionized water. Each bar represents the mean of two
independent determinations. (Inset) Transmission electron micro-
graphs of epidermal cells under the black and white stripes. Note
that only white stripe cells contain a large number of white gran-
ules composed mainly of uric acid.
Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle
FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1773
tion-dependent manner [21]. As expected, GBP activa-
ted TH and DDC gene expression only in the presence
of free Ca
2+
(Fig. 6B). Because we used a type of
EGTA that is not able to enter the cytosol [22], we
interpreted the results as suggesting that GBP stimu-
lates the epidermal cells to trigger Ca
2+
entry via cer-
tain Ca
2+
channels. Although we do not have
substantial data on GBP receptors and Ca
2+
channels
in the epidermal cells, it is reasonable to speculate that
activation of a GBP receptor opens a certain type of
Ca

2+
channel. Voltage-independent calcium channels
might contribute to GBP-induced Ca
2+
entry into the
epidermal cells [23]; furthermore, it is also possible that
the recently identified Ca
2+
release-activated Ca
2+
(CRAC) channels play a role in this system [24,25].
Monitoring cytoplasmic Ca
2+
concentration in the
cuticles revealed a significant difference, GBP increased
the Ca
2+
concentration in epidermal cells under the
black stripes, but not those under the white stripes,
suggesting that the GBP receptor population is higher
in the epidermal cells under the black stripes than
those under the white stripes. Preliminary experiments
of competitive receptor-binding assays using isolated
cuticles and
125
I-labeled GBP were carried out, but we
did not find any significant difference in the ligand-
binding capacities of black and white cuticles (data not
shown), indicating that the populations of GBP recep-
tors in the epidermal cells under both stripes are not

significantly different. Thus, we proposed another
mechanism by which DDC and TH genes are preferen-
tially expressed in the epidermal cells under the black
stripes.
It has been reported that uric acid is involved in
urinary stone formation; the presence of uric acid
increases the rate of stone growth from calcium salts
such as calcium oxalate and calcium phosphate in the
human kidney [26–28]. If a similar reaction proceeds in
the armyworm epidermal cells under the white stripes,
cytoplasmic free calcium ions could be decreased. As
partial confirmation of this, using laser confocal micr-
oscopy, Ca
2+
-induced fluorescence of Fluo-3 was not
seen in the cells under the white stripes of integument
that had been treated with GBP or calcium ionophore
(Fig. 7). We also showed that coincubation of CaCl
2
with uric acid decreased the free Ca
2+
concentration
in the incubation medium (Fig. 8). However, we were
unable to measure the cytoplasmic uric acid concentra-
tion in the epidermal cells because it is too difficult to
prepare a sample containing only the uric acid solubi-
lized in the cytoplasm. Therefore, the role of uric acid
in controlling Ca
2+
concentration in white stripe epi-

dermal cells should be carefully substantiated in the
future. We also cannot exclude the possibility that the
white stripe epidermal cells possess other Ca
2+
-buffer-
ing systems.
It will be interesting to determine the mechanisms
by which white stripe cells efficiently take up large
amounts of uric acid from the hemocoel. In mammals,
including humans, several types of urate transporter,
such as a voltage-sensitive urate transporter and a ura-
te ⁄ anion exchanger, have been identified [29–31].
Although the molecular mechanisms underlying urate
transport in insects are largely unknown, it is reason-
able to expect the presence of an active urate transpor-
ter in the plasma membrane of the epidermal cells
under the white stripes of the armyworm larvae.
In summary, two key enzymes in the dopamine syn-
thesis pathway, TH and DDC, are preferentially
expressed in the epidermal cells under the black stripes
on the armyworm larva cuticle. Expression of both
enzyme genes is enhanced by the insect cytokine, GBP,
via an increase of cytoplasmic Ca
2+
concentrations.
Our preliminary data showed that the epidermal cells
under the white stripes contain as many GBP receptors
just like the black stripe cells (data not shown). How-
ever, GBP-dependent enhancement of expression of
the two enzyme genes does not occur in the white

stripe cells. Although we do not have sufficient data to
explain this mechanism, one possible mechanisms is
that extremely high concentrations of uric acid
decrease the cytoplasmic Ca
2+
concentration, thereby
preventing expression of both enzyme genes in the cells
under the white stripes. As a consequence, melanin
synthesis proceeds only in the epidermal cells under
the black stripes, which produce the unique stripe pat-
tern in the cuticle of armyworm larvae.
Experimental procedures
Animals
Pseudaletia separata larvae were reared on an artificial diet
at 25 ± 1 °C in a photoperiod of 16:8 light ⁄ dark [10]. Pen-
ultimate instar larvae undergoing ecdysis between 4 and
4.5 h after starting the light period were designated as
day 0 last instar larvae.
Chemicals
l-(3,5-H
3
)Tyrosine was purchased from Amersham Bio-
science (Uppsala, Sweden). l-Tyrosine and A23187 (calcium
ionophore) were obtained from Nacalai Tesque Co.
(Kyoto, Japan) and Fluo-3 from Dojindo Laboratories
(Kumamoto, Japan), respectively. Grace’s insect cell culture
medium was purchased from Gibco-BRL (Rockville, MD).
All other chemicals were reagent grade.
Regulation of melanin synthesis in insect cuticle Y. Ninomiya and Y. Hayakawa
1774 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS

TH assay
Dissected tissue was homogenized in 100 lL of ice-cold
50 mm Hepes-KOH buffer (pH 7.0) containing 0.2 m
sucrose and 0.1% phenylthiourea by sonication (10 pulses
at 50 W). The homogenate was assayed directly for TH
activity using a slightly modified version of the method des-
cribed by Vie et al. [32]. The reaction mixture (total vol-
ume: 200 lL) consisted of 50 mm Hepes-KOH buffer
(pH 7.0), 1 mm dithiothreitol, 0.3 mm (6R)-5,6,7,8-tetra-
hydrobiopterin dihydrochloride, 10 lm ferrous ammonium
sulfate, 10 000 U catalase, 50 lml-tyrosine, 12.5 mCiÆ-
mmol
)1
l-(3,5-H
3
)tyrosine and the enzyme preparation.
The mixture, without (6R)-5,6,7,8-tetrahydrobiopterin dihy-
drochloride, was equilibrated at 37 °C for 5 min and after
adding (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride,
the reaction was performed for 30 min. After adding
600 lL of ice-cold 0.5 m trichloroacetic acid to stop the
reaction, the mixture was centrifuged at 20 000 g for
10 min at 4 °C. The supernatant was then transferred into
a microtest tube containing 120 mg of Norit A. The tube
was mixed occasionally for 30 min at 25 °C, and then cen-
trifuged at 20 000 g for 10 min at 4 °C. One hundred
microliters of the supernatant were transferred to a vial
with 1 mL of scintillation cocktail, and the radioactivity
was counted in a liquid scintillation counter (Aloka LSC-
5100, Tokyo, Japan).

Cloning and sequence analysis of TH cDNA
Total RNA was isolated from integuments of day 0 last
instar larvae using TRIzol reagent (Gibco-BRL) according
to the manufacturer’s instructions. Five micrograms of total
RNA was reverse transcribed with oligo(dT) primer using
ReverTra Ace (TOYOBO, Osaka, Japan). Degenerated
oligonucleotide primers were designed on the basis of
sequences of D. melanogaster and H. sapiens :5¢-TTYGCN
CARTTYWSNCARGA-3¢ and 5¢-TGRTCRTGRTANGG
YTGNAC-3¢. PCR cycling conditions were 35 cycles of
94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1.5 min.
PCR products were isolated and subcloned into the TA clo-
ning vector (pGEM-T Easy vector, Promega, Madison, WI)
and sequenced by a 310 DNA sequencer (ABI, Wellesley,
MA, USA). Full-length cDNA was isolated using the
RACE technique with a RACE system kit (Gibco-BRL).
Computer-assisted sequence analyses were performed by
genetyx-mac v. 10.0 (Software Development Co., Tokyo,
Japan).
RT-PCR
Two micrograms of total epidermal RNA was reverse
transcribed with oligo(dT) primer using ReverTra Ace
(TOYOBO). The cDNA was amplified with TH-specific
primer pair (5¢-CAGCTGCCCAGAAGAACCGCGAGA
TG-3¢, +11 to +36; and 5¢-GAACTCCACGGTGAACC
AGT-3¢, +1286 to +1305 bp), DDC-specific primer pair
(5¢-ATGGAGGCCGGAGATTTCAAAG-3¢, +1 to +22 bp;
and 5¢-ACGGGCTTTAAGTATTTCATCAGGC-3¢, +1405
to +1428 bp) and actin primer pair (5¢-TTCGAGCAG
GAGATGGCCACC-3¢ and 5¢-GAGATCCACATCTGYTG

GAAGGT-3¢). PCR was conducted under the following
conditions: 25 cycles at 94 °C for 1 min, 50 °C for 1 min,
and 72 °C 2 min.
Northern hybridization
Twenty micrograms of total RNA was separated on a 1%
formaldehyde–agarose gel and transferred onto a Hybond
N
+
nylon membrane. Hybridization was performed at
42 °C for 16 h in 50% formaldehyde containing 5· SSPE
and 0.5% SDS. The cDNA (nucleotides 11–1305) labeled
with [
32
P]dCTP was used as a probe. The membrane was
washed with 2· NaCl ⁄ Cit containing 0.1% SDS at 42 °C,
according to the protocols of Sambrook et al. [33]. Autora-
diogram was analyzed using a BAS-1500 imaging analyzer
(Fuji Film, Tokyo, Japan).
Production of polyclonal antibody
The cDNA fragment containing the ORF of P. separata
TH was cloned into pET32a (Novagen, San Diego, CA,
USA) and expressed as a recombinant protein in Escheri-
chia coli, BL21(DE3). Production of the protein containing
6 histidine-tag residues was induced by 0.4 mm isopropyl
thio-b-d-galactoside for 3 h at 37 °C. The recombinant pro-
tein was purified by a Chelating Sepharose Fast Flow col-
umn (Amersham Pharmacia Biotech, Piscataway, NJ)
charged with nickel. The purified protein was emulsified by
Titer Max Gold (CytRx Corporation, Los Angeles, CA,
USA) and injected into a rabbit to generate an anti-TH

IgG. Anti-TH IgG was precipitated by adding ammonium
sulfate to 40% saturation and further purified by an affinity
column of protein G–Sepharose (Amersham Bioscience).
Immunoblotting and immunocytochemical
analyses
Integuments dissected from larvae were homogenized in
80 mm Tris ⁄ HCl buffer (pH 8.8) containing 1% SDS and
2.5% 2-mercaptoethanol, and centrifuged at 20 000 g for
10 min at 4 °C. The supernatant was boiled for 5 min and
applied to a SDS ⁄ PAGE gel. Proteins separated by
SDS ⁄ PAGE were electrically transferred to a poly(vinylid-
ene difluoride) membrane filter, blocked and probed with
the primary antibody, anti-TH IgG. After washing thor-
oughly with 0.05% Tween 20 in Tris-buffered saline
(10 mm, 150 mm NaCl, pH 7.5), antigens were detected
Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle
FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1775
using peroxidase-conjugated secondary antibody and a
4-chloro-1-naphtol Immun-Blot Colorimetric Assay kit
(Bio-Rad Laboratories, Hercules, CA) [34].
Immunohistochemistry examination of integument sec-
tions was performed essentially as described by Somogyi &
Takagi [35], except that isolated tissues were fixed with 4%
paraformaldehyde in NaCl ⁄ P
i
(pH 7.4) for 2 h on ice. Anti-
gens were detected using HRP-conjugated anti-rabbit IgG.
In situ hybridization
The DIG-labeled TH RNA probe was prepared using the
Roche Biochemicals kit (Roche Molecular Biochemicals,

Indianapolis, IN). Hybridization and washing were carried
out as described previously [36].
Dissection and culture of integument
A whole abdominal integument (day 1 last instar larva) of
the test armyworm larva was dissected between the first
and second segments. Care was taken to remove all the
adhering fat body tissue from the integument. The dissected
integument was separated into dorsal and ventral parts.
After washing with NaCl ⁄ P
i
, the tissues were lightly blotted
with filter paper, weighed and immediately used for experi-
ments. Pieces of dorsal larval integument were cultured in
Grace’s medium with or without 1 nm GBP at 25 °C. As a
control, 1 n m BSA was added to the medium. To remove
extracellular free Ca
2+
, Grace’s medium containing 1 mm
EGTA was used. A23187 was dissolved in dimethylsulfox-
ide and added to the medium.
In vivo experiment
Total RNAs were extracted from the dorsal and ventral
integuments of day 1 last instar larvae 6 h after injection of
20 pmol of GBP. Control larvae were injected with 20 pmol
of BSA. RT-PCR was done as described above.
Confocal calcium imaging and electron
microscopy
A dissected dorsal integument (day 1 last instar larva) was
washed with Ca
2+

-free Carlson solution (120 mm NaCl,
2.7 mm KCl, 0.5 mm MgCl
2
, 1.7 mm NaH
2
PO
4
, 1.4 mm
NaHCO
3
, 2.2 mm glucose) and loaded with 10 lm Fluo-3
(Dojindo) at 25 °C for 30 min. After loading, the tissue was
washed twice with Ca
2+
-free Carlson solution, lightly blot-
ted with filter paper, and placed on a glass slide. The integu-
ment was stimulated by Grace’s medium with or without
1nm GBP and immediately excited with 488 nm wavelength
light using the confocal imaging system CellMap (Bio-Rad).
Transmission electron microscopy was carried out as des-
cribed previously [7].
Measurement of free Ca
2+
in uric acid solution
Uric acid suspension (70 mgÆmL
)1
) was incubated with
250 nm CaCl
2
at 25 °C. After incubation, the mixture was

centrifuged at 20 000 g for 10 min. The supernatant was
transferred to wells of 96-well assay plate and 500 nm Fluo-
3 solution was added to each well. The fluorescence inten-
sity was measured with a microplate reader, DTX880
(Beckman-Coulter).
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