Blue Biliprotein As an Effective Factor for Cryptic Colouration in Rhodinia

Journal of Insect Physiology 47 (20010) 205–212 www.elsevier.com/locate/jinsphys

Blue biliprotein as an effective factor for cryptic colouration in Rhodinia fugax larvae Hitoshi Saito * Department of Insect Genetics and Breeding, National Institute of Sericultural and Entomological Science, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan

Received 3 April 2000; accepted 30 June 2000

Abstract

The fifth instar larva of the saturniid silkworm, Rhodinia fugax, is light yellowish-green on its dorsal surface and dark green on the ventral surface with a lateral demarcation between the two colours. The larva of R. fugax closely resembles the leaves of the host plant, Quercus serrata, in colour and shape. The spectral reflectance of the larval integument of R. fugax corresponds to that of the Q. serrata leaf. In the larval integument, there is more blue biliproteins (BPs) on the ventral surface than on the dorsal surface. Light intensity influences larval colouration. The larval integuments are green under light conditions (1000 lux), whereas larvae kept in dark conditions (10 lux) turn yellow. The BP-I content of the haemolymph is also affected by light intensity. The quantities of BP-I and its blue chromophore are higher under light conditions than under dark conditions. In contrast, there is little difference in the yellow chromophore content between the two light intensities. When larvae are kept in the light, the BP-I content in the cuticle is higher than under dark conditions in both the ventral and dorsal surfaces, and its chromophore content parallels the BP content. However, the amounts of BP-II and its chromophore in the epidermis show no change with the light intensity. Moreover, the quantity of yellow chromophore in the integument is also not affected by light intensity. Therefore, light stimulates the accumulation of BP-I and its chromophore in the haemolymph and cuticle, whereas the accumulation of BP-II and its chromophore in the epidermis are not influenced by light intensity. These results suggest that BPs and their chromophores determine the larval colouration and may play an important role in the cryptic colouration of R. fugax larvae.  2000 Elsevier Science Ltd. All rights reserved.

Keywords: Biliprotein; Cryptic colouration; Biliverdin IXγ; Phorcabilin; Rhodinia fugax

1. Introduction Manduca sexta and characterised (Cherbas, 1973; Riley et al., 1984; Goodman et al., 1985; Holden et al. 1986, The caterpillars of phytophagous insects are fre- 1987). Ins is synthesised and stored in pigment granules quently green, and both the haemolymph and integument in the epidermis and secreted into both the haemolymph are often green. The green colouration presumably and the cuticle (Riddiford, 1982; Riddiford et al., 1990). serves as a protective camouflage (i.e. crypsis), helping The bilin binding protein of Pieris brassicae has been caterpillars to avoid predation. The colour results from crystallised (Huber et al., 1987a,b) and its complete a combination of blue (e.g. bilins: biliverdin IXγ, phorca- amino acid sequence has been determined (Suter et al., bilin and sarpedobilin) and yellow (e.g. carotenoids: β- 1988). Blue chromoproteins have also been isolated and carotene and lutein) pigments that are intimately associa- characterised from the larval haemolymph of several lep- ted with proteins (Kawooya et al., 1985; Kayser, 1985; idopteran insects: Heliothis zea (Haunerland and Bow- Law and Wells, 1989). ers, 1986), Trichoplusia ni (Jones et al., 1988), Spodop- The blue biliprotein insecticyanin (Ins) has been iso- tera litura (Yoshiga and Tojo, 1995), Agrius convolvuli lated from the haemolymph of the tobacco hornworm, (Saito and Shimoda, 1997), Attacus atlas (Saito, 1997) and Antheraea yamamai (Yamada and Kato 1989, 1991; Saito et al., 1998). In Samia cynthia ricini, a biliverdin- * Tel: +81-298-38-6096; Fax: +81-298-38-6028. binding protein (BBP) found in the moulting fluid during E-mail address: [email protected] (H. Saito). the larval–pupal ecdysis has been purified and character-

0022-1910/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S0022-1910(00)00115-3 206 H. Saito / Journal of Insect Physiology 47 (2001) 205–212 ised (Saito, 1993), and the N-terminal amino acid legs. The haemolymph was allowed to drip into ice- sequence of BBP has also been determined (Saito, 1994). cooled plastic micro-centrifuge tubes containing a few Recently, two BBPs were found in the larval haemo- crystals of 1-phenyl-2-thiourea, and then centrifuged at lymph of S. cynthia ricini (Saito, 1998a). 10,000g for 10 min at 4°C to remove the haemocytes. In Rhodinia fugax, blue biliproteins (BPs) are found The supernatant was used. in the haemolymph and integument of fifth instar larvae After collecting the haemolymph, the larvae were dis- (Saito, 1998b). The BP molecules from the haemolymph sected and the midgut, silk glands, fat bodies, and other and cuticle are assumed to be monomers, whereas the undesired tissues were removed with fine forceps. The epidermal BP is a dimer. The amino acid composition epidermis was collected with a spatula, and the cuticle and N-terminal amino acid sequences of the BPs from was washed with cold distilled water several times. Both the haemolymph and cuticle (BP-I) are very similar, but tissues were immediately transferred to plastic micro- the BP from the epidermis (BP-II) is quite different. R. centrifuge tubes that were chilled on a block of dry ice. fugax BPs consist of two different molecules. The blue The isolated epidermis and cuticle were stored at Ϫ20°C colour of BP is due to the presence of bile pigments, until used. which are non-covalently bound to an apoprotein. The Epidermis or cuticle (50 mg) was homogenised with blue pigments of BP-I and BP-II are different; BP-I con- 0.5 ml of 20 mM Tris–HCl buffer (pH 7.6) containing tains a phorcabilin-like pigment, while BP-II contains protease inhibitors (0.1 mM phenylmethyl sulfonyl flu- biliverdin IXγ. Furthermore, light stimulates the accumu- oride and 5 µg/ml soybean trypsin inhibitor) with 0.15 lation of bile pigments in the integument and cocoon, M NaCl. The precipitate was removed after centrifug- affecting the green colouration (Kato and Miyata, 1994). ation at 10,000g for 20 min at 4°C, and the supernatant In order to obtain information about the possible func- was used as the sample. After 20 mM Tris–HCl (pH 7.6) tion of BPs in the larval colouration of R. fugax, (1) the extraction, the precipitate was extracted with HCl:MeOH localisation of BPs in the integument and the relationship (5:95, v/v) solution. between BPs and larval colouration, and (2) the influence of light intensity on larval colouration, were exam- 2.4. Electrophoresis ined. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed on a 15% polyac- 2. Materials and methods rylamide gel containing 0.1% SDS (Laemmli, 1970). Electrophoresis was carried out at 30 mA until the brom- 2.1. Animals and food plants ophenol blue tracking dye reached the bottom of the gel. The gel was stained with Coomassie Brilliant Blue R- Eggs of R. fugax were obtained from wild-collected 250 (Fluka Chemie AG, Buchs). SDS–PAGE molecular female moths in Fukushima Prefecture. Larvae were weight standards (Low Range) were purchased from reared mainly on fresh oak leaves, Quercus serrata,at Bio-Rad Laboratories. 25°C under a natural photoperiod. The food plants were branches and leaves harvested from field grown trees. 2.5. Determination of BP concentration The branches were kept in tightly plugged vials of water. Fifth-instar larvae (5–7 days after last larval ecdysis) Each sample was separated on a 15% linear SDS– were used in the experiments. Animals were selected and PAGE. The gel was stained with Coomassie Brilliant their age determined according to live weight. Blue G-250 (Fluka Chemie AG, Buchs) and the BP band was scanned. The BP content was determined by NIH 2.2. Light irradiation Image (ver. 1.61). It was based on densitometry of the BP band and compared to a bovine serum albumin After the last larval ecdysis, some of the larvae were (BSA) band on the same gel. BSA was used as the stan- illuminated with light at one of two different intensities dard protein. [light (1000 lux) and dark (10 lux) conditions] under a photoperiod of 12 h light–12 h dark. White fluorescent 2.6. Absorbance spectrum tubes (Toshiba, FLR40S•W/M/36) were used as the light source, and light intensity was measured at the centre of The absorbance spectra of haemolymph and the the rearing room (2.4×2.4×2.8 m). extracts from the epidermis and cuticle were measured with a spectrophotometer (Beckman DU-650, USA). 2.3. Collection of haemolymph, epidermis and cuticle, and preparation of samples 2.7. Spectral reflectance

Haemolymph was collected from fifth-instar larvae The spectral reflectance of the larval integument of from incisions made by cutting off the abdominal pro- R. fugax and Q. serrata leaves were measured with a H. Saito / Journal of Insect Physiology 47 (2001) 205–212 207 spectrophotometer (Shimadzu MPC-3100, Kyoto) using vae of R. fugax resemble the leaves of the host plant of barium sulphate (Eastman Kodak Company, Rochester, Q. serrata in both colour and shape. NY) as a white reflectance standard. 3.2. Spectral reflectance of the larval integument and 2.8. Statistics the leaf of the host plant

Numerical data were expressed as the mean±SE. For Fig. 2 shows the spectral reflectance of the larval statistical analysis, Student’s t-test was used to compare integument of R. fugax and the leaf of its host plant, Q. results for the two light conditions. serrata. In the integument, the reflectance of the ventral surface was lower than that of the dorsal surface [Fig. 2(A)]. Both spectral reflectances had a small peak from 3. Results 540 to 669 nm. Similarly, in the leaf of the host plant, the reflectance of the adaxial surface was lower than that 3.1. Larval colouration of R. fugax of the abaxial surface and the spectral reflectance was similar to that of the larval integument [Fig. 2(B)]. These Fifth-instar larvae of R. fugax always hang from a results suggest that the spectral reflectances of the R. fugax branch [Fig. 1(A)]. The larvae are uniquely coloured in integument correspond with those of the Q. serrata leaf. two tones separated laterally. The dorsal surface is light yellowish green, whereas the ventral surface is dark green [Fig. 1(B)]. In addition, the body segments closely 3.3. Localisation of BP in the integument resemble indented leaf margins. The oak Q. serrata is one of the host plants of R. fugax [Fig. 1(C)]. The Fig. 3 shows the localisation of BP in the epidermis adaxial surface of its leaves are dark green, whereas the and cuticle by SDS–PAGE. Large amounts of BPs were abaxial surface are light green [Fig. 1(D)]. Thus, the lar- detected in the ventral epidermis (BP-II) and cuticle (BP-

Fig. 1. (A) Fifth-instar larva of Rhodinia fugax. (B) Lateral (upper), dorsal (middle), and ventral (lower) views of R. fugax larvae. (C) Leaves of Quercus serrata, the host plant of R. fugax. (D) Adaxial (left) and abaxial (right) views of Q. serrata leaves. 208 H. Saito / Journal of Insect Physiology 47 (2001) 205–212

Fig. 3. SDS–PAGE analysis of the presence of BP in the integument. Lane 1, molecular weight marker; lane 2, epidermis (ventral); lane 3, cuticle (ventral); lane 4, epidermis (dorsal); and lane 5, cuticle (dorsal). 10 µl of sample was applied to each lane. 6 µg of protein were loaded into lanes 2 and 4, and 10 µg were loaded into lanes 3 and 5. Arrow- heads indicate BP-I (open) and BP-II (solid).

Fig. 2. Spectral reﬂectance of the larval integument of R. fugax (A) and Q. serrata leaf (B). A: Ventral (———) and dorsal (- - -) surfaces of the integument. B: Adaxial (———) and abaxial (- - -) surfaces of the leaf.

I), while BP-I was not found in the dorsal epidermis (lane 4, Fig. 3). The BP contents of the epidermis and cuticle were compared on the ventral and dorsal surfaces (Fig. 4). The BP-I content of the ventral and dorsal cuticle was 1.40±0.09 and 0.98±0.09 µg/mg tissue weight, respectively. In the epidermis, the BP-II content in the ventral surface was 1.03±0.03 µg/mg tissue weight, whereas little was found on the dorsal surface (0.02±0.02 µg/mg tissue weight). The total amount of BPs (BP-I+II) on the ventral surface was larger than on the ventral surface. Therefore, the difference of larval body colour between Fig. 4. BP content in the epidermis and cuticle. (A) Ventral surface. the ventral and dorsal surfaces may result from the (B) Dorsal surface. Open and shaded columns indicate BP-I and BP-II, amount of BP in the integument. respectively. Values are the means±SE of ﬁve samples from different individuals. Tissue weight is the wet weight. All larvae had similar 3.4. Effects of light intensity on larval body colour body weights (8.95±0.16 g).

The effects of light intensity on the larval body colour 3.5. Effects of light intensity on the quantities of BP of R. fugax were examined under light and dark con- and chromophores in the haemolymph ditions (Fig. 5). When the larvae were reared under light conditions, the dorsal body was light yellowish-green The BP-I content in the haemolymph was compared and the ventral surface was dark green (upper, Fig. 5). at two different light intensities [Fig. 6(A)]. The BP-I In contrast, the dorsal body colour yellowed under dark content under light and dark conditions was 73.4±3.3 conditions (lower, Fig. 5). and 43.1±1.1 µg/ml, respectively. The amount of blue H. Saito / Journal of Insect Physiology 47 (2001) 205–212 209

and dorsal surfaces [Fig. 7(B)]. In the epidermis, the blue chromophore (absorbance at 669 nm) was only detected in the ventral surface, and it was more soluble in HCl:MeOH (5:95, v/v) than in 20 mM Tris–HCl (pH 7.6) buffer [Fig. 7(C)]. The total amount of blue chromophore in both sol- utions was essentially the same at the two light intensities. These results suggest that light stimulates the accumulation of BP-I and its chromophore in the cuticle, whereas the light intensity does not influence the accumulation of BP- II and its chromophore in the epidermis. Conversely, in the cuticle, the yellow chromophore (absorbance at 462 nm) was only extracted into 20 mM Tris–HCl (pH 7.6) buffer, and its content was not affec- Fig. 5. Effects of light intensity on the larval body colour of R. fugax. ted by the light intensity [Fig. 8(A)]. In contrast, in the Light (upper) and dark (lower) conditions. epidermis, the yellow chromophore (absorbance at 442 nm) was found only in the ventral surface and was extracted by HCl:MeOH (5:95, v/v) alone [Fig. 8(B)]. chromophore was measured as the absorbance maximum There was no significant difference between light and at 669 nm. The absorbance under light and dark con- dark conditions in the accumulation of yellow chromo- ± ± ditions was 1.63 0.05 and 1.04 0.14, respectively [Fig. phore in either tissue. 6(B)]. For the yellow chromophore, the absorbance spectrum of haemolymph was characterised by three absorbance maxima at 428, 453, and 483 nm. The 4. Discussion absorbance of the middle peak (453 nm) under light and dark conditions was 2.64±0.20 and 2.18±0.22, respect- 4.1. Cryptic colouration ively [Fig. 6(C)]. The caterpillar is the eating and growth stage in the 3.6. Effects of light intensity on the quantities of BP life cycle of a moth or butterfly. Survival in an environ- and chromophores in the integument ment filled with predators is difficult for a caterpillar. Predators include social vespid wasps, parasitic wasps, When the larvae were kept in light conditions, the BP- ants, spiders, and birds, and they cause immense damage I content in the cuticle was higher in both the ventral to caterpillar populations, with larval loss of 70–90%. and dorsal surfaces than in larvae kept in dark conditions Birds and parasitic wasps, which are the major predators, [Fig. 7(A)]. At each light intensity, the amount of BP-I can see colour. They have well-developed visual acuity was roughly the same in both surfaces of the cuticle. In and hunt by sight. As a countermeasure against pred- the epidermis, BP-II was found mainly in the ventral ators, caterpillars adopt several survival strategies, such surface, and the quantity was the same for both light as cryptic form and colouration, mimicry, and aposem- and dark conditions [Fig. 7(A)]. The blue chromophore atic colouration (Schmidt, 1990). (absorbance at 669 nm) in the cuticle was soluble in 20 The later instar larvae of many groups of Saturniidae mM Tris–HCl (pH 7.6) buffer, and there was more in are usually reverse countershaded; that is, the dorsal sur- light conditions than in dark conditions in both the ventral face, which faces downward in the resting position, is

Fig. 6. Effects of light intensity on the quantities of BP-I and chromophores in the haemolymph of R. fugax. (A) BP content, (B) blue chromophore, and (C) yellow chromophore. Open and shaded columns indicate light and dark conditions, respectively. Values are the means±SE of ﬁve samples from different individual. All larvae had similar body weights (7.62±0.15 g). Asterisks indicate signiﬁcantly different values: *pϽ0.05, **pϽ0.01. 210 H. Saito / Journal of Insect Physiology 47 (2001) 205–212

Fig. 8. Effects of light intensity on the quantity of yellow chromophore in the integument of R. fugax. (A) 20 mM Tris–HCl (pH 7.6) extract. (B) HCl:MeOH (5:95, v/v) extract after 20 mM Tris–HCl (pH 7.6) extraction. Open and shaded columns indicate light and dark conditions, respectively. Values are the means±SE of ﬁve samples from different individual.

2). It seems likely that the green colouration of R. fugax larvae is their major defence against caterpillar predators.

4.2. Blue BPs as an effective factor in camouflaging Fig. 7. Effects of light intensity on the quantities of BP and blue caterpillars chromophore in the integument of R. fugax. (A) BP content. (B) 20 mM Tris–HCl (pH 7.6) extract. (C) HCl:MeOH (5:95, v/v) extract In caterpillars, green pigments are observed in the after 20 mM Tris–HCl (pH 7.6) extraction. Open and shaded columns indicate light and dark conditions, respectively. Tissue weight is the layer of epidermal cells on the surface of the body, wet weight. Values are the means±SE of five samples from different where they play an important role in determining the individual. All larvae had similar body weights (7.62±0.15 g). Aster- visibility of caterpillars on their host plants. Blue bile pig- isks indicate significantly different values: ***pϽ0.001. ments, which give a bright green or blue colour, are fre- quently found in the haemolymph and integument. The bile lighter than the ventral surface. These larvae closely match pigments are usually associated with proteins such as BPs. the colour of the under surface of their host plant’s leaves. It is generally believed that the major function of blue BP The convex body segments of some genera (e.g. Antheraea is to provide camouflage for protecting the developing lar- and Actias) closely resemble indented leaf margins and vae (Kawooya et al., 1985; Law and Wells, 1989). compound leaves (Tuskes et al., 1996). In R. fugax, BPs have been efficiently isolated from In R. fugax, the cryptic colour and shape polymor- the haemolymph, epidermis, and cuticle of fifth instar phism of caterpillars appear to have evolved as strategies larvae (Saito, 1998b). There are usually two different to make the caterpillars appear similar to their host molecules: BP-I from the haemolymph and cuticle and plants. The larvae usually hang from a branch. The body BP-II from the epidermis. It is very interesting that the segments of the penultimate and final instar larvae phorcabilin-type pigment is distributed only in the hae- closely resemble the indented leaf margins of the host molymph and cuticle, whereas the biliverdin IXγ is plant. The spectral reflectance of the larval integument of found only in the epidermis. BP-I in the haemolymph R. fugax corresponds with that of the Q. serrata leaf (Fig. does not influence the larval body colour, so it probably H. Saito / Journal of Insect Physiology 47 (2001) 205–212 211 does not function as camouflage there. It is likely that Light-induced green colouration was found in the lar- BP-I is synthesised in the haemolymph, perhaps in hae- val haemolymph and integument of R. fugax (Figs. 6, mocytes, and secreted across the epidermis into the cuti- 7). The respective light/dark ratios of the BP-I and blue cle. It is necessary to determine whether this actually chromophore (absorbance at 669 nm) contents in the occurs. In Calpodes ethlius, the epidermis is involved haemolymph were 1.7 and 1.6 [Fig. 6(A, B)]. It seems with four routing classes of protein (Sass et al., 1993). likely that the amount of blue chromophore in the hae- It secretes peptides apically into the cuticle, basal into molymph changes in proportion to the change in the BP- the haemolymph, bidirectional, or transported to the cuticle I content with light irradiation. This phenomenon is also across the epidermis from the haemolymph (Locke et al., detected in the cuticle [Fig. 7(A, B)]. The light intensity 1994; Sass et al., 1993). A 66 kDa peptide is found abun- probably influences the syntheses of BP-I and phorca- dantly in cuticle and haemolymph but is made by spherulo- bilin-type pigment. These results suggest that light cytes and not by the epidermis (Sass et al., 1994). stimulates the accumulation of BP-I and its blue chromo- In this study, the amount of BPs (BP-I+II) in the ven- phore in the haemolymph and cuticle. However, it is an tral integument (epidermis and cuticle) was higher than open question whether the light-induced green colour- in the dorsal surface (Fig. 4). The quantity of BP-II in ation in this species comes from the synthesis of phorca- the dorsal epidermis was extremely low. The difference bilin-type pigment in the haemolymph and cuticle by in the larval colouration on the two surfaces results from phototransformation. In contrast to BP-I and its chromo- the amount of BPs in each surface of the integument. phore, BP-II and its blue chromophore (biliverdin IXγ) These results suggest that BPs may play an effective role in the epidermis are not influenced by light intensity. in the cryptic colouration of R. fugax larvae. Furthermore, the yellow component of R. fugax integument is a carotenoid (Kato and Miyata, 1994). Light 4.3. Effects of light intensity on larval colouration intensity has little effect on the accumulation of carotenoids in the R. fugax integument. Similarly, no change in yellow Three types of bilins (biliverdin IXγ, phorcabilin, and pigment content was observed in the accumulation of yel- sarpedobilin) have been reported in many lepidopteran low pigment in A. yamamai cocoons, although the yellow insects (Barbier, 1981; Kayser, 1985). Phorcabilin and pigment is not a carotenoid (Kato et al., 1989). Phytopha- sarpedobilin are obtained by irradiation of biliverdin IXγ gous insects generally do not synthesise carotenoids that with visible light in vitro (Barbier, 1981). However, the come from their food plants (Feltwell and Rothschild, process by which biliverdin IXγ is transformed into phor- 1974; Kayser, 1985). Therefore, it is possible that the cabilin and sarpedobilin in vivo is unknown. Moreover, the accumulation of carotenoids in a tissue is dependent on the biological significance of the transformation of biliverdin food irrespective of the light conditions. Consequently, the IXγ into phorcabilin and sarpedobilin is still unclear. phorcabilin-type pigment with its binding protein (BP-I) Colour changes and colour polymorphism modulated plays an important role in determining the shade of the by environmental factors, such as light, temperature, and green colouration of R. fugax larvae. humidity, have been found in many insects (Fuzeau- The R. fugax larva is a good model for studying the Braesch, 1985). In the Japanese oak silkworm, biological significance of bilin transformations. Further Antheraea yamamai, the larval haemolymph and cocoon experiments will be necessary to elucidate the mechanics are green, due to the presence of blue bilin and yellow of bilin transformations, such as whether light stimulates pigment. The green colouration of the cocoon is depen- the synthesis or accumulation of bilins. dent on the intensity of irradiation, whereas the larval haemolymph and integument are green irrespective of the light conditions (Kato et al., 1989). In spectrophoto- Acknowledgements metrical analyses, the bilins of the larval haemolymph The author thanks Dr T. Mitamura (Fukushima Seric- and integument differed from those of the cocoon (silk ultural Experiment Station) for providing R. fugax eggs. glands) and larval head capsule (Kato and Yamada, This study was supported by a National Institute Post 1991). The phototransformation of haemolymph bilin Doctoral Fellowship from the Japan Science and Tech- and its incorporation into the silk glands may be nology Corporation (JST). involved in the light-stimulated green colouration. Thus, light irradiation of the haemolymph effectively produces green colouration (Kato, 1991). In NMR analyses, the References chemical structure of bilin from the cocoon was similar to that of sarpedobilin (Yamada and Kato, 1994), which Barbier, M., 1981. The status of blue-green bile pigments of butterflies was isolated from Graphium sarpedon wings (Bois- and their phototransformations. Experientia 37, 1060–1062. Bois-Choussy, M., Barbier, M., 1977. Structure of sarpedobilin, a but- Choussy and Barbier, 1977). Moreover, similar light- terfly bile pigment. Experientia 33, 1407–1410. induced green colouration occurs in the larval integu- Cherbas, P., 1973. Biochemical Studies on Insecticyanin. Ph.D. thesis, ment and cocoon of R. fugax (Kato and Miyata, 1994). Harvard University. 212 H. Saito / Journal of Insect Physiology 47 (2001) 205–212

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