206 H. Saito Journal of Insect Physiology 47 2001 205–212
ised Saito, 1993, and the N-terminal amino acid sequence of BBP has also been determined Saito, 1994.
Recently, two BBPs were found in the larval haemo- lymph of S. cynthia ricini Saito, 1998a.
In Rhodinia fugax, blue biliproteins BPs are found in the haemolymph and integument of fifth instar larvae
Saito, 1998b. The BP molecules from the haemolymph and cuticle are assumed to be monomers, whereas the
epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from
the haemolymph and cuticle BP-I are very similar, but the BP from the epidermis BP-II is quite different. R.
fugax BPs consist of two different molecules. The blue colour of BP is due to the presence of bile pigments,
which are non-covalently bound to an apoprotein. The blue pigments of BP-I and BP-II are different; BP-I con-
tains a phorcabilin-like pigment, while BP-II contains biliverdin IX
γ . Furthermore, light stimulates the accumu-
lation of bile pigments in the integument and cocoon, affecting the green colouration Kato and Miyata, 1994.
In order to obtain information about the possible func- tion of BPs in the larval colouration of R. fugax, 1 the
localisation of BPs in the integument and the relationship between BPs and larval colouration, and 2 the influ-
ence of light intensity on larval colouration, were exam- ined.
2. Materials and methods
2.1. Animals and food plants Eggs of R. fugax were obtained from wild-collected
female moths in Fukushima Prefecture. Larvae were reared mainly on fresh oak leaves, Quercus serrata, at
25 °
C under a natural photoperiod. The food plants were branches and leaves harvested from field grown trees.
The branches were kept in tightly plugged vials of water. Fifth-instar larvae 5–7 days after last larval ecdysis
were used in the experiments. Animals were selected and their age determined according to live weight.
2.2. Light irradiation After the last larval ecdysis, some of the larvae were
illuminated with light at one of two different intensities [light 1000 lux and dark 10 lux conditions] under a
photoperiod of 12 h light–12 h dark. White fluorescent tubes Toshiba, FLR40S•WM36 were used as the light
source, and light intensity was measured at the centre of the rearing room 2.4
× 2.4
× 2.8 m.
2.3. Collection of haemolymph, epidermis and cuticle, and preparation of samples
Haemolymph was collected from fifth-instar larvae from incisions made by cutting off the abdominal pro-
legs. The haemolymph was allowed to drip into ice- cooled plastic micro-centrifuge tubes containing a few
crystals of 1-phenyl-2-thiourea, and then centrifuged at 10,000g for 10 min at 4
° C to remove the haemocytes.
The supernatant was used. After collecting the haemolymph, the larvae were dis-
sected and the midgut, silk glands, fat bodies, and other undesired tissues were removed with fine forceps. The
epidermis was collected with a spatula, and the cuticle was washed with cold distilled water several times. Both
tissues were immediately transferred to plastic micro- centrifuge tubes that were chilled on a block of dry ice.
The isolated epidermis and cuticle were stored at 220
° C
until used. Epidermis or cuticle 50 mg was homogenised with
0.5 ml of 20 mM Tris–HCl buffer pH 7.6 containing protease inhibitors 0.1 mM phenylmethyl sulfonyl flu-
oride and 5 µ
gml soybean trypsin inhibitor with 0.15 M NaCl. The precipitate was removed after centrifug-
ation at 10,000g for 20 min at 4
° C, and the supernatant
was used as the sample. After 20 mM Tris–HCl pH 7.6 extraction, the precipitate was extracted with HCl:MeOH
5:95, vv solution.
2.4. Electrophoresis Sodium dodecyl sulphate–polyacrylamide gel electro-
phoresis SDS–PAGE was performed on a 15 polyac- rylamide gel containing 0.1 SDS Laemmli, 1970.
Electrophoresis was carried out at 30 mA until the brom- ophenol blue tracking dye reached the bottom of the gel.
The gel was stained with Coomassie Brilliant Blue R- 250 Fluka Chemie AG, Buchs. SDS–PAGE molecular
weight standards Low Range were purchased from Bio-Rad Laboratories.
2.5. Determination of BP concentration Each sample was separated on a 15 linear SDS–
PAGE. The gel was stained with Coomassie Brilliant Blue G-250 Fluka Chemie AG, Buchs and the BP band
was scanned. The BP content was determined by NIH Image ver. 1.61. It was based on densitometry of the
BP band and compared to a bovine serum albumin BSA band on the same gel. BSA was used as the stan-
dard protein.
2.6. Absorbance spectrum The absorbance spectra of haemolymph and the
extracts from the epidermis and cuticle were measured with a spectrophotometer Beckman DU-650, USA.
2.7. Spectral reflectance The spectral reflectance of the larval integument of
R. fugax and Q. serrata leaves were measured with a
207 H. Saito Journal of Insect Physiology 47 2001 205–212
spectrophotometer Shimadzu MPC-3100, Kyoto using barium sulphate Eastman Kodak Company, Rochester,
NY as a white reflectance standard.
2.8. Statistics Numerical data were expressed as the mean
± SE. For
statistical analysis, Student’s t-test was used to compare results for the two light conditions.
3. Results
