Comprehensive microarray-based analysis for stage-specific larval camouflage pattern-associated genes in the swallowtail butterfly, Papilio xuthus
© Futahashi et al; licensee BioMed Central Ltd. 2012
Received: 21 March 2012
Accepted: 31 May 2012
Published: 31 May 2012
Body coloration is an ecologically important trait that is often involved in prey-predator interactions through mimicry and crypsis. Although this subject has attracted the interest of biologists and the general public, our scientific knowledge on the subject remains fragmentary. In the caterpillar of the swallowtail butterfly Papilio xuthus, spectacular changes in the color pattern are observed; the insect mimics bird droppings (mimetic pattern) as a young larva, and switches to a green camouflage coloration (cryptic pattern) in the final instar. Despite the wide variety and significance of larval color patterns, few studies have been conducted at a molecular level compared with the number of studies on adult butterfly wing patterns.
To obtain a catalog of genes involved in larval mimetic and cryptic pattern formation, we constructed expressed sequence tag (EST) libraries of larval epidermis for P. xuthus, and P. polytes that contained 20,736 and 5,376 clones, respectively, representing one of the largest collections available in butterflies. A comparison with silkworm epidermal EST information revealed the high expression of putative blue and yellow pigment-binding proteins in Papilio species. We also designed a microarray from the EST dataset information, analyzed more than five stages each for six markings, and confirmed spatial expression patterns by whole-mount in situ hybridization. Hence, we succeeded in elucidating many novel marking-specific genes for mimetic and cryptic pattern formation, including pigment-binding protein genes, the melanin-associated gene yellow-h3, the ecdysteroid synthesis enzyme gene 3-dehydroecdysone 3b-reductase, and Papilio-specific genes. We also found many cuticular protein genes with marking specificity that may be associated with the unique surface nanostructure of the markings. Furthermore, we identified two transcription factors, spalt and ecdysteroid signal-related E75, as genes expressed in larval eyespot markings. This finding suggests that E75 is a strong candidate mediator of the hormone-dependent coordination of larval pattern formation.
This study is one of the most comprehensive molecular analyses of complicated morphological features, and it will serve as a new resource for studying insect mimetic and cryptic pattern formation in general. The wide variety of marking-associated genes (both regulatory and structural genes) identified by our screening indicates that a similar strategy will be effective for understanding other complex traits.
KeywordsButterfly color pattern evolution expressed sequence tag (EST) larval body marking microarray Papilio polytes Papilio xuthus
Body coloration is an ecologically important trait that is often involved in prey-predator interactions through mimicry and crypsis (camouflage) . Because butterflies and moths spend most of their lives as larvae, which have soft bodies, they have developed a wide range of mechanisms to protect themselves from predators such as birds. The larval body markings of butterflies differ completely between closely related species and between individuals of the same species in different life stages [2, 3]. Despite the variety and significance of the larval color patterns, few studies have been conducted at a molecular level compared with the available research on adult butterfly wing patterns [4–9].
Spectacular changes in color pattern are observed in the caterpillar of the swallowtail butterfly Papilio xuthus. As a young larva, it mimics bird droppings (mimetic pattern), and during the final molting period, it switches to a green camouflage coloration (cryptic pattern). In addition, larvae in the final instar stage have a large eyespot on their thoracic segment that is believed to be useful for avoiding predation. One of the main factors involved in larval pattern formation is insect hormones. Previously, we revealed that larval pattern switch is regulated by juvenile hormone (JH) . The decline of JH titers on the first day of the fourth instar stage was the important factor controlling the formation of a green cryptic pattern in the fifth instar stage. The pattern transition occurs through ecdysis, and ecdysteroids appear to regulate the expression of several pigmentation genes. Topical application of 20-hydroxyecdysone alters the expression timing of several pigmentation genes [11, 12]. Comparing gene expression between stages and markings represents a promising strategy for identifying the genes responsible for larval pattern formation.
Previously, we found several pigmentation genes using a cDNA subtraction and candidate gene approach [10, 11, 13–16]. Six melanin synthesis (or associated) genes, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), yellow, tan, laccase2 and guanosine triphosphate cyclohydrolase I (GTP-CH I) are highly expressed in the presumptive black regions and ebony is highly expressed in the presumptive red region . We also reported that the combination of bilin-binding protein 1 (BBP1) and yellow-related gene (YRG) correlated perfectly with larval blue, yellow, and green coloration in three Papilio species [12, 17]. However, it was difficult to obtain novel marking-specific genes expressed at particular stages or genes with relatively low expression. For butterfly adult wing patterns, expressed sequence tag (EST) construction [18–20] and microarray analysis [6, 21] have been conducted to obtain marking-associated genes. Microarray analysis revealed that the expression of both a patterning gene (transcription factor gene optix) and effector gene (ommochrome synthesis gene cinnabar) are associated with red wing patterns in Heliconius species [6, 21].
In this study, we constructed an EST library of larval epidermis with more than 20,000 clones, and designed a microarray with the aim of comprehensively revealing the molecular mechanisms of larval mimetic and cryptic pattern formation. We performed microarray-based screening for marking-specific genes using six markings at 11 different stages, and verified the marking specificity of candidate genes by whole-mount in situ hybridization. We identified many novel marking-specific genes, including novel blue and yellow pigment-binding protein genes; a novel yellow family gene, the expression of which prefigures black cuticular markings; cuticular protein genes associated with marking-specific cuticular nanostructures; marking-associated regulatory genes; marking-specific ecdysteroid synthesis pathway genes; and Papilio-specific marking-associated genes. The data presented in this study provide a new resource to understand insect mimetic and cryptic pattern formation.
Results and discussion
Construction of a Papilioexpressed sequence tag database
Summary of P. xuthus, P. polytes and B. mori expressed sequence tag data sets
Bombyx mori a
Sequenced clone numbers (EST numbers)
Number of clones corresponding to ribosomal RNA
Number of clones corresponding to mitochondrial DNA
Number of putative genes (including isoforms)
Summary of P. xuthus gene set
P. xuthus genes (total)
P. xuthus genes with coding sequences (> 50 amino acids)
with homology to Danaus plexippus proteins (P < 1e-10)
with homology to Bombyx mori proteins (P < 1e-10)
with homology to Drosophila melanogaster proteins (P < 1e-10)
Comparison of highly expressed genes among two Papilio species and Bombyx
Microarray-based screening for marking-specific genes in P. xuthus
To determine whether we could compare microarray data across all samples, we first compared the normalized signal intensity of housekeeping genes (ribosomal protein genes). We found that these genes were ubiquitously expressed as expected, and their normalized signal intensity among the samples was within two-fold of their average values in most cases (Additional files 7 and 8). We also checked several melanin synthesis genes, for which we have previously reported their stage-specific and marking-specific expressions, and confirmed the expected specific expression (see below). Thus, we considered that a comparison of the normalized signal intensity across all of the examined samples is reliable for screening of the novel marking- and stage-specific genes.
Identification of several candidates for blue and yellow pigment-binding proteins
Because JHBP has high ligand specificity [37, 38] and it has been assumed to be monomeric in solution , PCBP1 and PCBP2 may bind to different types of carotenoids. Other takeout/JHBP genes (for example, Px-0150 and Px-0591) also had marking specificity and were enriched in the fourth instar (Figure 6A), suggesting that these genes also have a supporting role in carotenoid binding. Because the presence of several carotenoids, including alpha-carotene, beta-carotene and lutein, have been reported for P. xuthus , these takeout/JHBP family genes of P. xuthus may each recognize a different carotenoid.
Notably, the locust carotenoid-binding protein (red asterisk in Figure 6B) did not form a single cluster with Px-0019 or Px-0220, which is similar to the case of BBP, suggesting that both blue and yellow pigment-binding proteins evolved convergently among insect species. Green coloration among lepidopteran larvae appears to have emerged independently in the phylogenetic tree . The independent occurrence of BBPs and carotenoid-binding proteins within the lipocalin family genes and takeout/JHBP family genes may reflect the convergent evolution of larval green coloration.
Identification of the novel marking-specific melanin synthesis gene, yellow-h3
Unexpectedly, yellow-f3 (Px-4077) and yellow-f4 (Px-0903) of P. xuthus were not upregulated in black markings, but instead were CG-enriched (Figure 7A). Conversely, yellow-h3 (Px-0430) was upregulated in black markings, and it exhibited a very similar expression pattern to that of yellow (Figure 7A). Using whole-mount in situ hybridization, we confirmed that the spatial expression pattern of yellow-h3 prefigured the black markings similar to yellow (Figure 7C). The only previous report concerning the role of yellow-h3 in coloration was the demonstration of its association with black regions in Heliconius wings by RT-PCR, but its spatial expression patterns have not been clarified . Our data, combined with the Heliconius results, suggest that yellow-h3 functions in melanin biosynthesis in lepidopteran species (Figure 7D) similarly to yellow-f and yellow-f2 in Drosophila.
Based on our microarray analysis, three yellow family genes, yellow-f3, yellow-h2 and yellow-e, were regarded as CG-enriched genes (Figures 3 and 7A). yellow-f3 was highly expressed during the late molting period similar to TH and DDC, and yellow-h2 and yellow-e were highly expressed during the middle of the molting period similar to yellow (Figure 7A), implying that these genes may function in inhibiting melanin pigmentation. In the silkworm B. mori, yellow-e disruption promoted melanin pigmentation in the larval head and tail, where strong yellow-e expression was detected , which is consistent with our results. In melanin synthesis, it is assumed that arylalkylamine-N-acetyltransferase (NAT) activity is involved in synthesis of N-acetyldopamine, a precursor of colorless cuticle [5, 45, 46]. However, disruption of NAT in B. mori resulted in an overall blackish phenotype only in the adult stage, whereas it had little effect on larval pigmentation [46, 47]. Our EST datasets did not contain NAT or NAT-like genes, suggesting that NAT gene is not the primary factor for colorless cuticular production in larval stages. Because green is the color of the epidermis seen through the colorless cuticle, the observation of CG-enriched yellow family genes (CG in Figure 3) implies that yellow family genes are major negative regulators of melanin pigmentation in the larval stage instead of the NAT gene (Figure 7D). Taxa-specific gene duplication found between Drosophila and lepidopteran species (for example, yellow-f, yellow-h, Figure 7B), and the reverse function implicated for yellow-f family genes between Diptera and Lepidoptera suggest that the function of the yellow gene family has diversified among insect taxa.
Marking-specific cuticular protein genes
Candidates for marking-associated patterning genes
E75A and E75B are involved in the ecdysteroid signaling cascade, and their expression is induced by ecdysteroids . We therefore examined the effect of ecdysteroids by the topical application method  and found that higher E75 expression is maintained in black eyespot regions (Figure 9B; probes were designed to target common regions of E75A and E75B). The expression pattern of E75 coincided with the eyespot pattern, which was similar to yellow , suggesting that E75 had a marking specificity in the early molting stage. In Manduca sexta, both E75A and E75B are involved in the stage-specific gene expression of DDC both directly and indirectly . Our results suggest that E75A and/or E75B regulate both the marking specificity and stage specificity of several black marking-associated genes. As we described previously, several cuticular protein genes also exhibited marking specificity and stage specificity (Figure 8). Although it is still not clear how color and nanostructure are determined, one possible explanation is that marking-specific transcription factors involved in the ecdysteroid signaling cascade regulate both pigmentation and cuticular protein genes. E75 is one strong candidate mediator of the hormone-dependent coordination of larval pattern and nanostructure formation.
Although spalt and E75 were the only two transcription factor genes for which we were able to detect marking-associated expression by whole-mount in situ hybridization, microarray analysis suggested that E(spl) region transcript mbeta and fringe, both involved in the Notch signaling pathway, also have marking specificity. In the butterfly wing pattern, Notch is involved in intervein markings , and fringe is upregulated by 20-hydroxyecdysone . The Notch signaling pathway may also be involved in hormone-dependent pattern formation in butterfly larvae.
The marking-specific ecdysteroid biosynthesis enzyme gene, 3-dehydroecdysone 3b-reductase
Papilio-specific marking-associated genes
Among 2,746 P. xuthus genes (ORF > 50 amino acids), more than 400 genes had no sequence similarity with the genome sequences of other insects, including silkworm and monarch butterfly (Additional file 5). Through microarray analysis and whole-mount in situ hybridization, we found three Papilio-specific genes (that is, has no sequence similarity with known proteins including the monarch butterfly Danaus, Bombyx genome sequences, and the Butterfly Base sequence database  with clear marking specificity (Px-0559, Px-3233 and Px-3244), and one gene, Px-0011, with only low sequence similarity with the monarch butterfly (that is, butterfly specific). Px-0559, Px-3233 and Px-3244 expression coincided with black, yellow and eyespot markings, respectively (Figure 10). Px-0011 expression was also strongly associated with black markings (Figure 10). These proteins have no known domains and display no similarity to any characterized proteins. Px-0011 and Px-3233 have signal peptide sequences, and Px-0011, Px-0559 and Px-3233 are threonine-, arginine-, and tyrosine-rich proteins, respectively (Additional file 1). These genes were expressed during the middle or late stages of the molting period, suggesting that these genes were structural proteins. Although the precise function of these genes is currently unknown, this is the first example of species-specific genes associated with pattern formation.
In this study, we succeeded in obtaining a large-scale catalog of marking-specific genes and stage-specific genes from the swallowtail butterfly larvae by microarray analysis based on EST datasets. We confirmed the marking specificity for transcription factors, hormone-related genes, cuticular protein genes, pigment synthesis and binding genes, and novel Papilio-specific genes by whole-mount in situ hybridization. The marking-specific genes identified in this study indicated that the molecular mechanisms of insect pigmentation are likely to be both conserved and diversified across insect taxa. For example, several of the melanin synthesis genes with clear marking specificity in Papilio species (Figure 7) [12, 16] have been reported to be responsible for the larval color mutants of the silkworm B. mori [16, 58, 59], as well as associated with the cuticular pigmentation of various insect orders [5, 45, 60–62], which suggests their conserved role in cuticular pigmentation across insect taxa. Conversely, our results also indicate that genes that have several paralogs in one species tend to diversify their function across insect taxa, such as yellow family genes, lipocalin family genes and takeout/JHBP family genes (Figures 5, 6, 7). We also found that there were several Papilio-specific genes with clear marking specificity, suggesting that species-specific genes contribute to marking formation. Notably, some of the regulatory genes involved in larval pattern appeared to be different from those of adults (for example, Distal-less involvement in eyespots was less obvious in larvae), whereas others appeared to participate in both larval and adult pattern formation (for example, spalt expression associated with black markings). Furthermore, we found that both the transcription factor E75 and the ecdysteroid synthesis enzyme 3DE 3b-reductase had clear marking specificity, suggesting their involvement in ecdysteroid-dependent coordinated gene regulation in larval pattern formation. Ecdysteroid is also involved in butterfly adult wing pattern , which suggests the possibility that these genes contribute to pattern formation in the adult wing. The wide variety of marking-associated genes identified by our screening indicates that the strategy utilized in this study is effective for clarifying pattern formation in species without genome information. The gene collection and the expression profile presented in this study will be invaluable for exploring not only Papilio but also insects in general.
Experimental animals and developmental staging
P. xuthus was either purchased from Eiko-Kagaku (Osaka, Japan), kindly provided by Dr. Akira Yamanaka (Yamaguchi University, Japan), or collected from the field. Larvae were reared on leaves of Zanthoxylum ailanthoides (Rutaceae) at 25°C under long-day conditions (16 h light and 8 h dark). The staging of the molting period was based on the time when HCS occurred, as well as spiracle and hair pigmentation .
Construction and sequencing of the cDNA library
Total epidermal RNAs of P. xuthus and P. polytes were isolated from 50 individuals per species during the molting period (35 individuals at third molt and 15 individuals at fourth molt) using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. After the attached muscle and fat body were removed, whole dorsal integuments from the thoracic 2 segment to the abdominal 7 segment were dissected from larvae. Total RNAs were mixed and subjected to cDNA library construction.
The cDNA library construction was carried out by the Dragon Genomics Center (Takara Bio Inc., Mie, Japan) by the following procedure. The mRNA was purified using an Oligotex-dT30 Super mRNA purification Kit (Takara, Bio Inc., Mie, Japan), and then cDNA libraries were constructed using a cDNA synthesis Kit (Stratagene, La Jolla, CA, USA). First-strand cDNA synthesis was carried out with oligo d(T)18 primers. Synthesized cDNA were size-selected and ligated into the EcoRI and XhoI sites of pBluescript II SK(+) vector (Stratagene). The ligated products were then transformed into competent DH10B Escherichia coli cells by electroporation. The transformed cells were plated onto Luria Broth media and incubated overnight.
For sequencing of cDNA libraries, white colonies were randomly selected and sequenced from both ends using an ABI prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), which was carried out at National Institute of Genetics (Shizuoka, Japan) with the support of the MEXT Genome Support Project for the P. xuthus cDNA library, and at the National Institute of Agrobiological Sciences (Ibaraki, Japan) for the P. polytes cDNA library. All EST sequences have been deposited [DDBJ: FY174038-FY210626, FY302525-FY358875]. Among these ESTs, sequences corresponding to ribosomal RNA [DDBJ: AB674749] and mitochondrial DNA [DDBJ: EF621724] were eliminated. The remaining ESTs were subjected to cluster analysis using a Phred/Phrap/Consed software package . After automatic clustering, we checked every sequence manually for each alignment, and divided and reassembled the putative chimeric sequences. SignalP 3.0  was used for signal peptide prediction.
Microarray experiments and data analysis
The Papilio 15 K oligo-microarray slide (60-mer oligonucleotides on 15,208 spots, Agilent Technologies, Palo Alto, CA, USA) was constructed using 8,000 Papilio unique sequences (3,376 from P. xuthus, 4,612 from P. polytes and 12 from P. machaon). We could not design an appropriate probe for 42 genes because of low sequence complexity (indicated by 'n.e.' in Additional file 1). Probe sequences were designed using Agilent's web portal eArray , and one or two probes each for P. xuthus, P. machaon and P. polytes gene were constructed (probe sequences of P. xuthus are shown in Additional file 7).
For the microarray experiment of mimetic pattern, total epidermal RNAs of six developmental stages (0, 4, 8, 11, 16 hours after HCS of the third molting period and intermolt (day 2) of the third instar) were isolated from white (abdominal 2 to 4 segments) and black (abdominal 4 and 5 segments) regions independently (see Figure 2, left). For the microarray experiment of cryptic pattern, total epidermal RNAs of five developmental stages (1, 4, 10, 13 hours after HCS of the fourth molting period and intermolt (day 2) of the fourth instar) were isolated from the thorax (thoracic 2 segment with no markings), eyespot (thoracic 3 segment), abdomen (abdominal 3 and 4 segments with no markings), and V-shaped marking (abdominal 4 and 5 segments) regions independently (see Figure 2, right).
We performed two color microarray hybridizations. Mimetic white, cryptic thorax and cryptic abdomen were independently labeled with cyanine 3-CTP (Cy3), and mimetic black, cryptic eyespot, and cryptic V-shaped markings were independently labeled with cyanine 5-CTP (Cy5) in all stages. Double-stranded cDNA and labeled cRNA were synthesized using the Low RNA Input Linear Amplification Kit according to the manufacturer's instructions (Agilent Technologies). Total RNA (400 ng each for Cy3 and Cy5) was converted to cDNAs with T7 promoter oligo-dT primer and Moloney Murine Leukemia Virus reverse transcriptase. Second-strand cDNA was transcribed to cRNA with T7 RNA polymerase and 10 mM Cy3 or Cy5. The labeled cRNA was purified using an RNeasy Mini Kit (Qiagen, Tokyo, Japan).
Hybridization was performed using an In situ Hybridization Kit Plus (Agilent Technologies). One microgram each of Cy3-labeled cRNA and Cy5-labeled cRNA were mixed, fragmented and hybridized to each oligo-microarray at 65°C for 17 h at 4 rpm. The arrays were washed in 6 × saline sodium citrate, 0.005% TritonX-102 for 10 min at room temperature and in 0.1 × saline sodium citrate, 0.005% TritonX-102 for 5 min at 4°C. Intensities of the hybridized probes were detected with an Agilent G2565BA Microarray Scanner with 10 mm scan resolution, and the signals were extracted with G2565AA Feature Extraction Software v.7.1 (Agilent Technologies). Normalization was performed 'per spot' and 'per chip' using the GENESPRING program. The low expression level spots in all cells were removed (fluorescence intensity is approximately lower than 100). Microarray data were deposited in the Gene Expression Omnibus [GEO:GSE37920]. The normalized fluorescence intensity of all samples and each probe sequence are shown in Additional file 7.
We used microarray-based screening to obtain the genes involved in the overall steps of pattern formation, with the following strategies: comparing gene expression between different markings to identify marking-specific genes; comparing gene expression between mimetic and cryptic patterns to identify instar-specific genes; and checking the gene expression profile during the molting periods to identify stage-specific genes. To do this, we dissected larval epidermis at 11 different stages (Figure 2, red arrowheads) for microarray screening. Because cryptic patterns are assumed to be determined in the JH-sensitive period (green box in Figure 2) , six different stages before the JH-sensitive period were expected to be associated with the mimetic pattern and five different stages after the JH-sensitive period were analyzed for genes associated with cryptic pattern. For the mimetic pattern, we separately dissected the white region (w) and the black region (b), whereas for the cryptic pattern, we separately dissected the thoracic 2 segment (T), eyespot (E), abdomen region with no markings (A) and V-shaped markings (V) (Figure 2). Expression profiles of 11 developmental stages or six markings (mimetic white, mimetic black, cryptic thorax, cryptic eyespot, cryptic abdomen and cryptic V-shaped marking) were grouped by self-organizing maps with GenePattern software . This algorithm is often used to summarize microarray data. We obtained expression profiles in terms of relative expression rather than absolute expression levels by dividing the average expression levels of each gene.
Alignment was conducted based on translated protein sequences using Clustal W program implemented in MEGA5, and phylogenetic trees were constructed by the Neighbor joining method with the MEGA5 program . The confidence of the various phylogenetic lineages was assessed by the bootstrap analysis.
Whole-mount in situhybridization
Whole dorsal integuments from the thoracic 2 segment to the abdominal 7 segment were dissected from larvae. After the body fat and muscle attached to the epidermis were carefully removed, the larval epidermis was fixed immediately in 4% paraformaldehyde in PBS (137 mM NaCl, 8.10 mM Na2HPO4, 2.68 mM KCl and 1.47 mM KH2PO4, pH 7.4). Whole-mount in situ hybridization was performed as described previously [13, 15–17]. An RNA probe for each gene was prepared using the DIG RNA Labeling Kit (Roche Biochemicals, Mannheim, Germany). A color reaction for digoxigenin-labeled antisense RNA probes was performed at room temperature in 100 mM Tris-HCl, 100 mM NaCl and 50 mM MgCl2 (pH 9.5) containing 3.5 μL/mL 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt and 4.5 μL/mL nitroblue tetrazolium chloride. Digoxigenin-labeled sense strand probes were used as negative controls.
Scanning electron microscopy analysis
Cuticular nanostructure of eyespot and V-shaped marking region was observed by scanning electron microscopy using a Hitachi TM-1000 scanning electron microanalyzer (Hitachi High-Technologies Corp., Tokyo, Japan).
- 3DE 3b-reductase:
- DDC :
expressed sequence tag
- GTP-CH I :
guanosine triphosphate cyclohydrolase I
head capsule slippage
juvenile hormone-binding protein
open reading frame
putative carotenoid-binding protein
reverse transcriptase polymerase chain reaction
- TH :
- YRG yellow-related gene :
We are very grateful to Dr. Asao Fujiyama for arranging the EST project, to Dr. Akira Yamanaka for kindly providing many larvae of P. xuthus, to Dr. Tetsuya Kojima for help with scanning electron microscopy analysis, to Dr. Yuki Nakamura for help with the microarray analysis, to Mrs. Kyoko Chagi for technical support of in situ hybridization, and to Dr. Mizuko Osanai-Futahashi for her critical reading of the manuscript. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas "Comparative Genomics" from the Ministry of Education, Culture, Sports, Science and Technology of Japan 20017007 (to HF), Grants-in-Aid for Scientific Research "Genome Science" 221S0002 (to HF), Grants-in-Aid for Scientific Research 22128005 (to HF), and Grant-in-Aid for JSPS fellows 07J03511 (to RF).
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