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

A GBP-induced increase in cytoplasmic Ca2+ was seen in epider-mal cells under the black stripes but not those beneath the white stripes, suggesting that a difference in Ca2+ concentratio

regulator of melanin-synthesis enzymes in armyworm

larval cuticle

Yosuke Ninomiya1and Yoichi Hayakawa2

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: hayakayo@cc.saga-u.ac.jp

(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

Ca2+, 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 Ca2+ was seen in epider-mal cells under the black stripes but not those beneath the white stripes, suggesting that a difference in Ca2+ 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 Ca2+ and hence decrease its free concentration, we discuss the possibility that uric acid, as well as GBP, contributes to the difference in cytoplasmic Ca2+within the epidermal cells

Abbreviations

DDC, Dopa decarboxylase; Dopa, 3,4-dihydroxy- L -phenylalanine; GBP, growth-blocking peptide; TH, tyrosine hydroxylase.

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

Ca2+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 activactiv-ity 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

0 2 4 6

Dorsal Ventral

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

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.

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 Ca2+-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 Ca2+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 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 Ca2+ 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 Ca2+ was higher

Met

219 376

Epidermis form of PsTH

Stop 1884 531

219

Met

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

rRNA

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.

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

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

M

m 1

A S

M

n

1 M

m

mM

M

n Ca l C P

G GTA

E 2 P

G

M

M

n

P

G GTA E

20pmol BSA

20pmol GBP

C

in vivo 6h after injection

TH

DDC

Actin

in vitro 6h incubation

A23187 0µ M A23187 50µ M

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.

dependent expression of the two enzyme genes in the

dorsal integument requires cytoplasmic Ca2+

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 Ca2+ concentrations

specifically in the epidermal cells under the black

stripes

We monitored the Ca2+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 Ca2+ 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 Ca2+ 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 Ca2+ 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)

c)

a)

c)

e)

B

Fig 7 Ca2+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.

and ventral epidermal cells (Fig 8, insert) [7] The

effect of uric acid on soluble calcium ion

concentra-tions was examined by coincubating CaCl2 with uric

acid We found that incubation with > 70 mgÆmL)1

uric acid significantly decreased the free Ca2+

concen-tration (Fig 8) The decrease in free Ca2+ occurred

in a time-dependent manner: free Ca2+ 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 presdiffer-ence 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 Ca2+-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 Ca2+ concentration and the expression levels of both enzymes A calcium ionophore (A23187) elevated expression of both enzyme genes, suggesting that Ca2+

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 Ca2+ 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

Ca2+ influx into brain synaptosomes in a

Time (min)

2µm

h

Wit t s

e ri e p

a l

Bc

e p i s 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 n M ) was incubated with 70 mgÆmL)1uric

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.

tion-dependent manner [21] As expected, GBP

activa-ted TH and DDC gene expression only in the presence

of free Ca2+ (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 Ca2+entry via

cer-tain Ca2+ channels Although we do not have

substantial data on GBP receptors and Ca2+channels

in the epidermal cells, it is reasonable to speculate that

activation of a GBP receptor opens a certain type of

Ca2+ channel Voltage-independent calcium channels

might contribute to GBP-induced Ca2+ entry into the

epidermal cells [23]; furthermore, it is also possible that

the recently identified Ca2+ release-activated Ca2+

(CRAC) channels play a role in this system [24,25]

Monitoring cytoplasmic Ca2+ concentration in the

cuticles revealed a significant difference, GBP increased

the Ca2+ 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 and125I-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, Ca2+-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 CaCl2

with uric acid decreased the free Ca2+ 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 Ca2+ 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 Ca2+ -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 Ca2+ 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 Ca2+ 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 ± 1C 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-H3)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

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 lm l-tyrosine, 12.5

mCiÆ-mmol)1 l-(3,5-H3)tyrosine and the enzyme preparation

The mixture, without (6R)-5,6,7,8-tetrahydrobiopterin

dihy-drochloride, was equilibrated at 37C 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 4C The supernatant was then transferred into

a microtest tube containing 120 mg of Norit A The tube

was mixed occasionally for 30 min at 25C, and then

cen-trifuged at 20 000 g for 10 min at 4C 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

94C 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 94C for 1 min, 50 C for 1 min, and 72C 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

42C for 16 h in 50% formaldehyde containing 5· SSPE and 0.5% SDS The cDNA (nucleotides 11–1305) labeled with [32P]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 37C 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 4C 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

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⁄ Pi(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⁄ Pi, 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 25C As a

control, 1 nm BSA was added to the medium To remove

extracellular free Ca2+, 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 Ca2+-free Carlson solution (120 mm NaCl,

2.7 mm KCl, 0.5 mm MgCl2, 1.7 mm NaH2PO4, 1.4 mm

NaHCO3, 2.2 mm glucose) and loaded with 10 lm Fluo-3

(Dojindo) at 25C for 30 min After loading, the tissue was

washed twice with Ca2+-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

1 nm 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 Ca2+in uric acid solution Uric acid suspension (70 mgÆmL)1) was incubated with

250 nm CaCl2at 25C 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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