- Research article
- Open Access
Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development
© Connahs et al. 2016
- Received: 30 June 2015
- Accepted: 14 March 2016
- Published: 31 March 2016
Butterfly wing color patterns are an important model system for understanding the evolution and development of morphological diversity and animal pigmentation. Wing color patterns develop from a complex network composed of highly conserved patterning genes and pigmentation pathways. Patterning genes are involved in regulating pigment synthesis however the temporal expression dynamics of these interacting networks is poorly understood. Here, we employ next generation sequencing to examine expression patterns of the gene network underlying wing development in the nymphalid butterfly, Vanessa cardui.
We identified 9, 376 differentially expressed transcripts during wing color pattern development, including genes involved in patterning, pigmentation and gene regulation. Differential expression of these genes was highest at the pre-ommochrome stage compared to early pupal and late melanin stages. Overall, an increasing number of genes were down-regulated during the progression of wing development. We observed dynamic expression patterns of a large number of pigment genes from the ommochrome, melanin and also pteridine pathways, including contrasting patterns of expression for paralogs of the yellow gene family. Surprisingly, many patterning genes previously associated with butterfly pattern elements were not significantly up-regulated at any time during pupation, although many other transcription factors were differentially expressed. Several genes involved in Notch signaling were significantly up-regulated during the pre-ommochrome stage including slow border cells, bunched and pebbles; the function of these genes in the development of butterfly wings is currently unknown. Many genes involved in ecdysone signaling were also significantly up-regulated during early pupal and late melanin stages and exhibited opposing patterns of expression relative to the ecdysone receptor. Finally, a comparison across four butterfly transcriptomes revealed 28 transcripts common to all four species that have no known homologs in other metazoans.
This study provides a comprehensive list of differentially expressed transcripts during wing development, revealing potential candidate genes that may be involved in regulating butterfly wing patterns. Some differentially expressed genes have no known homologs possibly representing genes unique to butterflies. Results from this study also indicate that development of nymphalid wing patterns may arise not only from melanin and ommochrome pigments but also the pteridine pigment pathway.
- Polyphenol Oxidase
- Butterfly Wing
- Yellow Gene
- Wing Development
Arguably, among the most striking examples of morphological variation are the stunning array of colors and patterns that decorate the wings of butterflies. The spectacular diversity of butterfly wing patterns has been shaped by natural selection to serve a variety of adaptive functions, ranging from mate recognition and courtship to predator avoidance and deterrence [1–3]. Although many of the ecological processes shaping color patterns are well documented, the underlying molecular and developmental program generating these patterns still remains largely unknown [1, 4, 5].
Over the past two decades, research has revealed that genes involved in wing color pattern development also belong to an ancient gene regulatory network (GRN) for wing construction [2, 6, 7]. This network has been proposed to serve as a pre-patterning template for downstream pigment genes [1, 8, 9]. Studies on wing development in Drosophila melanogaster, ants and aphids have characterized expression patterns of this gene regulatory network [10, 11]; however, no comprehensive analysis has been conducted in butterfly wings.
In addition to regulating wing development, many of these selector genes and morphogens appear to have been redeployed in novel developmental contexts to specify wing color patterns, indicating a potential co-option event [1, 18–21]. Eyespots are the most well studied wing color pattern elements with at least 12 genes identified in the focus and colored rings [3, 19]. In nymphalid butterflies, expression of antennepedia, en, sal, dll and notch is observed in the focus of the eyespot [3, 19]. Many of these same wing developmental genes are also expressed in other pattern elements [18, 22]. These studies reveal a remarkably diverse role of these genes in controlling wing size and shape and also development of wing color patterns.
Wing color patterns are determined when each scale cell specifies a particular color pigment. A number of pigment pathways described in Drosophila have also been identified in butterflies including ommochromes (red, yellow and orange-- found only in nymphalids), and the melanins (black, brown and tan) [18, 23–25]. In general, ommochrome pigments appear earlier in pupal wing development than melanin pigments . While many of the genes involved in pigmentation are well characterized, the connection between the developmental genes in the wing GRN and pigmentation pathways remains unclear [9, 27]. A link has been established between developmental genes and specific pigments; for example, en has been mapped to the ring of gold scales around the eyespots of Bicylcus [3, 19, 22]. Melanin pigmentation has also been shown to be associated with sal expression in pierid butterflies  and wntA signaling in Heliconius butterflies [4, 29, 30]. These examples implicate a role for patterning genes in regulating downstream pigment genes; however, identifying the gene networks and regulatory mechanisms linking the initial patterning process to final scale pigmentation remains an important challenge.
Next generation sequencing has become a valuable tool for surveying the transcriptome of non-model organisms . Lepidoptera are a diverse order of insects, and there are still relatively few well annotated genomic resources . Our current understanding of the genes involved in wing color pattern development is based on a small selection of species, primarily Junonia coenia, Bicyclus anynana and members of Heliconius [3, 27, 33–35]. A diversity of species should be examined to better understand how wing color patterning has evolved in butterflies. Here, we conduct a transcriptome analysis to examine the temporal dynamics of genes expressed during wing color pattern development in the nymphalid butterfly Vanessa cardui, with a specific focus on genes involved in patterning, pigmentation and gene regulation.
Vanessa cardui caterpillars and artificial diet were purchased from Carolina Biological Supply Company (Burlington, NC). The caterpillars were reared individually at ambient temperature (~28 °C). Wing discs were dissected from caterpillars at two developmental time points in the final instar; early 4th larval (EL) and late 4th larval (LL) stages representing 2 and 4 days post-molt respectively, and at three time points during pupal development, early pupa (EP) 2 days, pre-ommochrome (PO) 5 days and late melanin (LM), 8 days post-pupation. Prior to harvest, larvae were weighed. The thorax, including the first abdominal segment, was harvested and placed immediately in RNAlater® (Ambion) and stored at 4 °C for at least 48 h prior to dissection. Pupal wings were dissected from live pupa using a Zeiss Stemi-2000 microscope and placed immediately in RNAlater and stored at 4 °C. Imaginal wing discs (fore and hind wings) were carefully dissected from the larva and placed in RNAzol® RT (Molecular Research Center Inc.) for RNA isolation. For pupal wing samples, fore and hind wings were placed in RNAzol for RNA isolation. All tissues were weighed and processed using an electric homogenizer followed by RNA isolation using isopropanol. Concentration of RNA was measured using a ND-1000 spectrophotometer (NanoDrop products, Wilmington, DE) (A260/A280 > 1.8) and integrity was assessed using electrophoresis on a formaldehyde-agarose gel. The RNA samples were diluted in water to a concentration of 25 ng/μl in 50 μl. All fore and hind wing discs were pooled for each larva prior to RNA extraction. RNA from 5 individual larvae was diluted and pooled for each developmental time point (in total four biological replicates of 5 pooled individuals per time point). A total of 11 larval libraries were prepared for RNA sequencing and transcriptome assembly. Two control libraries (one from early 4th instar and one from late 4th instar) were used for downstream expression analyses. The remaining libraries were part of a separate study involving treatment manipulations and were excluded from differential expression analyses. Following RNA isolation of pupal wings, forewings and hindwings were pooled for each individual and the RNA diluted as described above. For the 2 and 5-day pupal wings, diluted RNA from 4 individuals was pooled. Diluted RNA from 3 individuals was pooled for the 8-day time point. Two biological replicates of pooled samples were prepared for each pupal time point.
Illumina sequencing and de-novo transcriptome assembly
Library construction was performed using the Illumina TruSeq RNA Sample Preparation Kit v2 (University of Utah Microarray and Genomic Analysis Core Facility). Briefly, total RNA (100 ng to 4 ug) was poly-A selected using poly-T oligo-attached magnetic beads. The Poly-A RNA was eluted from magnetic beads, fragmented and primed with random hexamers. First strand cDNA synthesis was performed using Superscript II Reverse Transcriptase (Invitrogen) and then converted to blunt-end fragments with an A-base following second strand synthesis. Adapters containing a T-base overhang were ligated to the A-tailed DNA fragments. The ligated fragments were PCR-amplified (12 cycles) and the amplified library purified by Agencourt AMPure XP beads (Beckman Coulter Genomics). Concentration of the amplified library was measured with a NanoDrop spectrophotometer. To determine the size distribution of the sequencing library an aliquot was resolved on an Agilent 2200 Tape Station. Quantitative PCR (KapaBiosystems Kapa Library Quant Kit) was used to calculate the molarity of adapter ligated library molecules and the concentration of the libraries was adjusted to a concentration of 10 nM. Library concentration was further adjusted in preparation for analysis on the Illumina HiSeq 2000 platform.
De-novo transcriptome assembly was performed using 50 bp raw reads in CLC Genomics Workbench (v. 6.5.1) with a word size of 40. The parameters were modified throughout the assembly and mapping process to optimize similarity (e.g. 0.96–0.98) and length fraction (e.g. 0.4–0.98). To control for assembly of chimeric sequences, contigs with read coverage less than 20 were selected at each step of the assembly for BLASTX searches against the nr database in NCBI. All sequences with less than 20 reads were discarded if BLASTX searches revealed potential chimeras. Mismatch, insertion and deletion costs were set at 2, 3 and 3 respectively.
CLC Genomics (v. 8.0) was used to identify differentially expressed transcripts by mapping reads from each library to the entire transcriptome assembly. The edgeR bioconductor package available in v. 8.0 was used to perform statistical analyses of read count data using TMM normalization . Comparisons were made between LL vs. EP, (larval to early pupa) EP vs. PO (early pupa to pre-ommochrome) and PO vs. LM (pre-ommochrome to late melanin). The RNA-seq data were filtered to obtain transcripts ≥ 500 bp for reliable annotation and with a False Discovery Rate (FDR) p-value <0.001 for each comparison. This FDR cut-off allowed us to obtain highly significant results and a manageable number of sequences for annotation.
Sequence annotation was performed on the filtered sequences using a variety of approaches including local BLASTX (E value < 1 x10-5) to Drosophila melanogaster peptide database (FlyBase.org). Transcripts with no blast hits to D. melanogaster were annotated in BLAST2GO PRO by performing a BLASTX search against the entire non-redundant database at NCBI (E value < 1 x10-5). Following annotation, the transcripts were processed through the BLAST2GO pipeline .
To identify regulatory genes (transcription factors) and genes involved in pigmentation, gene names and symbols were obtained from the Animal Transcription Factor Database (bioinfo.life.hust.edu.cn/AnimalTFDB using Drosophila melanogaster) and Amigo2 (amigo2.geneontology.org) and matched to annotated contigs using header extractor (users-b0irc.au.dk/biopv/php/fabox) and the VLOOKUP function in Excel. Heatmaps were generated in JMP (v. 11.0) (SAS Institute Inc., Cary, NC) to visualize expression patterns of pigment associated genes and transcription factors using Wards distance measure and z-score normalization of RPKM values.
Drosophila wing gene regulatory network
For the candidate gene approach, genes from the Drosophila wing gene regulatory network were identified following a BLASTX search against the Drosophila peptide database. To examine the temporal expression patterns of these genes across all development stages, a two-way ANOVA was conducted in JMP, using transformed RPKM values. To normalize gene expression levels we included the glutamate receptor as a covariate (i.e., as an internal control). The glutamate receptor was identified as an internal control by filtering the transcriptome data for transcripts with consistent expression levels across all developmental stages (FDR p > 0.2).
Comparison of butterfly transcriptomes
We identified homologous sequences from Vanessa cardui in other available butterfly wing transcriptomes including Heliconius melpomene maletti, Heliconius melpomene cythera (InsectBase) , Junonia coenia (datadryad.org)  and also the genome of Danaus plexippus (MonarchBase) . The transcriptome comparison was conducted by first obtaining unigenes for the V. cardui transcriptome using CD-Hit suite  with similarity set to 0.95. CD-Hit clustered all sequences with similarity ≥95 % and retained only the longest transcript thereby removing splice variants and reducing redundancy. Each butterfly transcriptome/peptide database was compared to V. cardui unigenes using TBLASTX and BLASTX (for peptides in D. plexippus) (1E-10-5). Venny 2.0.2 (bioinfogp.cnb.csic.es/tools/venny/) was used to visualize results and identify transcripts common to all butterflies. We also searched for homologs of V. cardui unigenes in the genome of the silk moth Bombyx mori (Silkdb.org)  (BLASTX) and also the brain transcriptome from Bicyclus anynana  (TBLASTX) using the same parameters.
Quantitative PCR validation
An independent experiment was designed to validate the transcriptome results. Wing discs and pupal wings were dissected at the same developmental stages as the transcriptome study with seven biological replicates per stage. RNA isolation was performed as described above. RNA quality was checked for degradation on a formaldehyde-agarose gel. A qPCR was also performed on the RNA with primers for the glutamate receptor to confirm absence of any genomic DNA contamination. cDNA synthesis was performed with an iScript kit (BioRad) in a single run for all samples using 1 μg of input RNA (20 μl reaction). An aliquot of cDNA was diluted to the equivalent of 2 ng total RNA input/μl for qPCR. Primers were designed for the following genes: wg, sal, en, dll, ddc, pale, ebony, tan, vermillion, kf, and cinnabar (Additional file 1: Table S1). We used cDNA (2 ng/μl) from wing samples to amplify PCR products using Accuzyme™ 2x reaction mix (Bioline). Glutamate receptor was selected as a housekeeping gene based on results from whole transcriptome data. The PCR products were checked for a single band (75 bp) on a 1 % agarose gel, purified using a Thermo Scientific purification kit and quantified using Nanodrop. Standard curves were generated using an initial concentration of 2 picograms of PCR product and serial 10-fold dilutions . qPCR was performed using 2 μl of cDNA template with Evagreen Supermix (BIO-RAD) (10 μl reaction/well), and run on a CFX384 Real time system (Bio-rad C1000 Thermocycler) with the following conditions 95 °C 30s, 95 °C 5 s, 60 °C 5 s for 40 cycles. A bivariate regression analysis was performed in JMP to compare expression patterns for the RNA-seq and qPCR data for all 12 genes listed in Additional file 1: Table S1.
Availability of data and materials
Raw reads used to assemble the transcriptome are deposited in the short read archive at NCBI under Bioproject accession PRJNA284000. Larval libraries used for expression analyses are listed under accession numbers SRX1603967 and SRX1603981. Pupal libraries used for expression analyses are listed under accession numbers SRX1605764 SRX1605766, SRX1605767, SRX1605768, SRX1605769 and SRX1605770.
The annotated transcriptome is available at InsectBase, www.insect-genome.com/query.php?accession=IBVcarT00001, accession number IBVcarT00001.
This research did not require any permits to obtain the butterflies and does not involve any endangered or protected species.
Summary of de-novo transcriptome assembly
Total size of transcriptome
Total number of reads
Mean no. reads for early 4th larval libraries (EL)
Mean no. reads for late 4th larval libraries (LL)
Mean no. reads for 2 day pupal libraries (EP)
Mean no. reads for 5 day pupal libraries (PO)
Mean no. reads for 8 day pupal libraries (LM)
Total number of contigs
Number of contigs >200 bp
aMean contig length
aMedian contig length
aMax contig length
15, 506 bp
1, 266 bp
Number of contigs ≥ 500 bp
Number of unigenes
Of the 89, 069 contigs, a handful of contigs were identified that exhibited constant levels of expression across all developmental stages. Following BLAST searches, we identified one of these contigs as a putative glutamate receptor. The remaining contigs that exhibited constant expression did not match any known sequences on NCBI and are likely non-coding RNA. Quantitative PCR confirmed that expression of the glutamate receptor did not vary across developmental stages (p > 0.05). This gene was used as a covariate for subsequent qPCR analyses.
Correlation of qPCR and RNA-seq data
Top differentially expressed transcripts
Genes showing the highest fold change between LL vs. EP included genes from the Osis family, cuticular and ecdysone induced proteins. Transcripts most strongly down-regulated included aldehyde oxidases, larval serum and cuticle proteins. From EP to PO stages, the aldehyde oxidases were significantly up-regulated as were a number of cuticular proteins and chitinases. One of the most strongly down-regulated genes at this stage was E75 (Ecdysone inducible protein 75). During the PO to LM transition, the ammonium transporter (Amt) exhibited the largest fold change increase and several aldehyde oxidases were strongly down-regulated. For all stages there were many uncharacterized transcripts that were significantly up or down regulated. These transcripts exhibited the largest fold change for both the EP vs. PO and PO vs. LM stages (Additional file 3: Table S2 and Additional file 4: Table S3 lists all DE genes and GO annotations respectively).
Drosophila wing gene regulatory network
We next examined whether any of the genes from the Drosophila wing GRN were included in the top differentially expressed transcript list (FDR p < 0.001). Both en and hh were significantly down-regulated from the larval to early pupal stage. Hedgehog and wg were also significantly down regulated during the transition from the early pupal to pre-ommochrome stage. There was no evidence of differentially expressed genes during the pre-ommochrome to late melanin stages. Other genes known to be important for wing development in Drosophila and potentially wing patterning in butterflies were also significantly down-regulated, including Notch, Wnt6 and Wnt10 (Additional file 3: Table S2). Though not differentially expressed, antennepedia and aristalless were observed in the transcriptome of V. cardui. We were unable to identify optix, a gene strongly associated with red pigmentation in Heliconius butterflies [27, 44].
Pigment genes differentially expressed during wing development
Transcription factors differentially expressed during wing development
Comparison of butterfly transcriptomes
Here, we describe the first transcriptome analysis of wing development in the painted lady butterfly, Vanessa cardui and examine expression dynamics of genes involved in tissue patterning, pigmentation and gene regulation. One goal of this study was to compare expression patterns of genes from the wing GRN in Drosophila with genes involved in pigmentation. Many genes from the Drosophila wing GRN are expressed in butterfly wing patterns suggesting these developmental genes may function as a pre-patterning template for genes involved in pigmentation [9, 25]. The temporal expression patterns of these genes have not been examined in butterfly wings thus it remained unclear how their expression corresponds with the timing of pigment genes. If patterning genes are involved in regulating pigmentation, their expression may be significantly up-regulated at some stage during wing color pattern development. For example, expression of spalt and distal-less are associated with melanin pigmentation [28, 47]; therefore, upregulation of these genes may coincide with increased expression of melanin genes.
Contrary to our expectation, we did not observe these wing-patterning genes significantly up-regulated at any stage during pupation. In fact, many of these genes were significantly down-regulated during pupal development. Expression of these patterning genes generally peaks during larval and early pupal stages, subsequently declining during wing development. Any potential role these genes play in regulating pigmentation likely occurs during these earlier developmental stages, long before pigmentation becomes visible on the wing. Our results also revealed that genes characterized in the Drosophila wing GRN (with the exception of abd-A) are also expressed in the wings of V. cardui. Many of these genes have been identified in the wings of ants and pea aphids [10, 11] and more recently Bombyx mori . Whether this wing GRN is functionally conserved in these insects is currently unknown.
Largest number of transcripts upregulated during the EP to PO transition
To further explore genes involved in wing development we examined the top differentially expressed transcripts between late larval and early pupal stages (LL vs EP), early pupal to pre-ommochrome (EP vs. PO) and pre-ommochrome to late melanin stages (PO vs LM). We found that the highest number of up-regulated transcripts occurred during the EP to PO stage indicating that this is a dynamic period during wing development. At this stage, white scale patches are clearly visible on the wing and ommochrome pigments are deposited shortly after. The transition from LL to EP stages was marked by a larger turnover of transcripts compared to the other pupal stages, which shared a greater proportion of genes in common. This is likely due to significant morphological changes occurring between the larval and pupal wings. As reported in B. mori, we also observed an increasing number of transcripts down-regulated during the progression of wing development . Therefore the process of wing maturation appears to be largely regulated by gene silencing.
Temporal dynamics of pigment gene expression
As expected, an increasing number of pigment-associated genes were up-regulated during wing development. We predicted that if patterning genes regulate pigment genes, we should observe temporal co-expression of these genes during wing color pattern development. As patterning genes peak in expression during larval and early pupal stages, expression of some pigment genes should also be up-regulated at this time. Surprisingly, genes involved in the melanin pathway (yellow-f2, yellow, yellow-h, ple) were significantly up-regulated during the LL to EP transition, almost a week before melanin pigments become visible on the wing. However, not all melanin genes were up-regulated; the enzyme Dopa-decarboxylase (Ddc) was significantly down-regulated along with genes associated with the melanin immune response . Melanin pigment is produced when tyrosine is converted by pale (tyrosine hydroxylase) to dopa and further to dopamine by Ddc . Polyphenol oxidases (PO) and enzymes from the yellow gene family are thought function downstream to convert dopa and/or dopamine to black pigment. There are contrasting views regarding whether black melanin is derived primarily from dopa or dopamine, although recent RNAi experiments of melanin genes indicate that dopamine is a necessary precursor for black pigmentation . Our results suggest that the enzyme specifically required for melanin pigmentation is repressed during early pupation. Interestingly, Dat, the enzyme responsible for producing colorless cuticle was significantly up-regulated  along with Megalin (mgl) a multi-ligand receptor which regulates cuticle integrity and localization of yellow . Loss of Dat in B. mori and mgl in Drosophila results in ectopic melanin pigmentation, highlighting the critical role of these genes in restricting melanization [52, 53]. Taken together, these results suggest that partial up-regulation of the melanin pathway is required for pre-patterning wing regions fated for melanization. Alternatively, these genes could be involved in other developmental processes during early pupation such as cuticle development [54–56].
We also examined whether any homologs of pigment granule genes were significantly up-regulated in addition to the up-regulation of pigment genes. In Drosophila these genes are involved in the biogenesis of pigment granules which house either the brown ommochromes or red drosopterins [57, 58]. Although we identified several putative pigment granule genes (claret, garnet, ruby, dor) in the wing transcriptome, only deep orange (dor) was significantly up-regulated (during early pupation). The other granule genes were not differentially expressed at any developmental stage. It still remains unclear whether the model proposed for eye pigmentation in Drosophila is consistent with the process of scale pigmentation, as pigment granules have not been identified in butterflies [25, 27].
During the pre-ommochrome stage prior to red pigmentation, many genes involved in ommochrome synthesis (brown, yellow, red pigments) were strongly down-regulated including cinnabar, scarlet, white and kfase. These results support earlier studies indicating that expression of most genes in the ommochrome pathway are up-regulated during larval wing development and decline during pupation . For both ommochrome and melanin genes, there is a significant time lag between the up-regulation of pigment genes and onset of pigment synthesis in the wings. This lag suggests that other genes may also be involved in regulating pigmentation. Examination of spatial patterns of gene expression in pupal wings might resolve this apparent discrepancy in timing between peak transcript levels and pigmentation. Another possibility is that pigmentation enzymes are regulated post-translationally .
We observed significant up-regulation of pteridine enzymes involved in Drosophila red eye pigmentation, xanthine dehydrogenase (rosy), maroon-like, (mal), and phenylalanine hydroxylase (henna) coincident with down-regulation of ommochrome genes [59, 60]. Pteridines are well described in the Pieridae, with different pterins responsible for white, yellow and orange pigments due to variation in absorption of violet and UV light [61, 62]. Pterins are also cofactors for ommochrome biosynthesis in pigment cells of ommatidia in Drosophila as well as enzymes involved in growth and differentiation . Whether the pteridine pathway serves any functional role during wing pigmentation in nymphalid butterflies is currently unknown. A recent study revealed that rosy is up-regulated in the red morph of Junonia coenia implicating involvement of both pteridine and ommochrome pathways in the development of red pigmentation .
Transition from the early pupa to pre-ommochrome stage was also characterized by significant up-regulation of all the major genes involved in the melanin pathway including several members of the yellow family. Although expression of genes from the melanin pathway is strongly associated with black pigmentation, some of these genes are also expressed in red and yellow wing tissues in Heliconius and Papilio butterflies [27, 64]. Up-regulation of melanin and pteridines during the pre-ommochrome stage may indicate involvement of these pathways in non-melanic pattern elements in V. cardui.
As expected, most genes significantly up-regulated during the late melanin stage were those from the melanin pathway, although the most strongly up-regulated gene was black which functions in suppressing melanin synthesis [65, 66]. Increased expression of black along with Dat and ebony may prevent melanization in non-black regions of the wing as shown in Heliconius, where ebony and Dat1 are up-regulated in red and yellow tissues respectively . We also observed strong up-regulation of yellow-d2, which increased almost 200-fold in expression. In Heliconius, yellow-d is up-regulated in red tissues, revealing that not all yellow genes are associated with black pigmentation . Whether yellow-d and yellow-d2 share similar functions in pigmentation is currently unknown. The function of yellow genes in pigmentation is poorly understood as the same paralog can exhibit contrasting functions in different species; for example, yellow-d is associated with melaninized tissue in Bombyx mori  and unmelanized tissue in Heliconius species [27, 68]. Interestingly, in V. cardui, yellow genes strongly down-regulated during the pre-ommochrome stage (yellow-f2 and yellow-d2) were significantly up-regulated during the late melanin stage, while the opposite trend was observed for yellow-y, yellow-e and yellow-h. Collectively, these results suggest different functional groups of yellow genes, which may play either activating or repressive roles in melanin pigmentation.
Transcription factors differentially expressed during wing development
To explore candidate genes that may potentially regulate scale color fate we identified differentially expressed transcription factors during wing color pattern development. During early pupation and scale determination , we observed significant up-regulation of shaven/sparkling and polis au dos (pad). In Drosophila, shaven/sparkling represent mutations in two distinct enhancers of D-Pax2 that not only influence eye morphology (sparkling), but also control development of sensory bristles of the pupal retina (shaven) . Pad is also involved in bristle development and appears to function as a negative regulator of achaete-scute . It has been proposed that bristles and scale cells are homologous structures based on shared expression of achaete-scute . Although achaete-scute was not differentially expressed during early pupation, up-regulation of bristle development genes supports the assertion that these structures are homologous.
Ovo/shavenbaby, a gene involved in oogenesis, epidermal differentiation and trichome formation , was also among the most significantly up-regulated transcription factors. In Heliconius butterflies, ovo is differentially expressed in hindwings, indicating a possible role of this gene in color patterning . This appears to be the case in Drosophila where ovo regulates expression of yellow during the development of pigmented denticles . Interestingly, we also observed significant up-regulation of yellow-y during the early pupal stage. Whether ovo is also involved in regulating expression of yellow or other pigment genes in butterfly wings is currently unknown.
During the pre-ommochrome stage, we found that many up-regulated transcription factors have generalized roles in cell migration, growth and differentiation (slow border cells (slbo), pebbled/hindsight (peb), bunched (bun) and delta (Dl)). The specific function of these genes in the context of butterfly wing development has not been investigated; therefore, we can only speculate on their potential roles. Interestingly, several of these genes interact with the signaling molecule Notch. The Notch receptor has been implicated as an important regulatory molecule for specifying butterfly wing patterns [74, 75], although the specific signaling pathway leading to pattern development is still unknown. It has been proposed that slbo/bun/Notch may function as a conserved signaling cassette in regulating cell fate boundaries. In Drosophila, this signaling cassette specifies anterior/posterior identity of follicle cells during oogenesis [45, 76]. Bun also functions in restricting Notch activity to the wing margin, producing a notched wing phenotype when mutated . Our finding that bun, slbo and dl were significantly up-regulated during the PO stage suggests this signaling cassette may also be conserved in Lepidoptera, however we did not observe differential expression of Notch. We did identify eight putative splice variants of Notch. Several of these were significantly down-regulated during the other pupal stages. If these genes are part of a conserved Notch signaling cassette, then activation during the pre-ommochrome stage may specify positional information of pattern elements just prior to visibility on the wing (approximately 24 h later).
Transcription factors involved in hormonal regulation
Hormones play a fundamental role regulating insect metamorphosis and can also influence pigmentation of butterfly wing pattern elements [38, 77, 78]. Ecdysone signaling begins with the release of ecdysone (20E). Ecdysone (20E) is a ligand for the ecdysone receptor and a heterodimer of EcR (Ecdysone Receptor) and Usp (Ultraspiracle) . Hormone regulation of gene expression occurs when the heterodimer binds ecdysone response elements in the promoters of ecdysone-responsive target genes . In V. cardui, we observed contrasting patterns of expression for EcR and ecdysone-responsive genes (E75C, Eip93F, Eip74EF, Eip78C). EcR expression peaked during the pre-ommochrome stage and declined during the late melanin stage; however, the opposite pattern was observed for ecdysone-responsive genes. Previous work demonstrates that EcR can have a repressive function when ecdysone levels are reduced . Thus, ecdysone levels may decline during the pre-ommochrome stage, resulting in EcR repression of these target genes. Studies on pigmentation in lepidopteran larvae suggest that expression of melanin and ommochrome genes are regulated by ecdysone-induced transcription factors [79, 82, 83]. Work in Drosophila also shows that Ddc contains an ecdysone response element (EcRE) that binds EcR . Other ecdysone inducible genes, like E75, have been proposed as potential regulators of Ddc during larval cuticle development in Manduca sexta . There are comparatively few studies examining ecdysone regulation of pigment genes during pupal development, therefore the potential role of these transcription factors is largely unknown. Further investigation is required to identify whether other pigment genes also possess EcRE’s, and if ecdysone-inducible transcription factors regulate their expression during wing color pattern development.
We have assembled the first wing transcriptome for Vanessa cardui and identified a suite of genes involved in patterning, pigmentation and gene regulation, including many genes not previously described in butterflies. Some of these genes include transcription factors, which were significantly up-regulated during wing development. These factors may be involved in regulating wing color pattern development. In addition to ommochrome and melanin genes, we identified genes from the pteridine pathway, indicating that nymphalids may utilize this pathway for generating pigments. Our analysis also identified genes from the Drosophila wing GRN; genes from this network show similar temporal expression dynamics to enzymes involved in the ommochrome pathway which peak early during wing development. Although some melanin genes were also up-regulated during this developmental stage, Ddc, which is required for melanin pigmentation was significantly down-regulated. These results suggest that the melanin pathway is repressed during early pupation; however, up-regulation of some melanin genes indicates functionality in aspects other than pigmentation. Finally, our comparative analysis of transcriptomes across butterfly species identified a common set of transcripts with no known homologs to other animals and may represent novel genes unique to Lepidoptera.
We gratefully acknowledge funding support to HC and RBS from NSF ND EPSCoR grant #EPS-0184442, and TR NSF EPSCoR grant #EPS-0814442 and ND EPSCoR State Funding project #UND0017937, a graduate women in science fellowship (Adele Lewis Grant Fellowship) to HC, University of North Dakota Biology Department Wheeler award and University of North Dakota School of Graduate Studies Summer Research Stipend to HC. We also thank Kasey Chelemedos and Whitni Redman for providing outstanding assistance in butterfly rearing. KC was supported by an undergraduate NSF AURA award through grant #EPS-0814442, project #UND0018877. WR was supported through the NSF REU Site Grant 0851869 by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103442. We would like to thank the Office of the Vice President of Research, University of North Dakota for assistance in publication costs. We are also grateful to Muhammad Luqman Aslam and Nesibe Özsu for advice on bioinformatic analyses and two anonymous reviewers for their suggestions on improving the manuscript.
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- Beldade P, Brakefield PM. The genetics and evo-devo of butterfly wing patterns. Nat Rev Genet. 2002;3:442–52.PubMedGoogle Scholar
- Brakefield PM, French V. Butterfly wings: the evolution of development of colour patterns. Bioessays. 1999;21:391–401.View ArticleGoogle Scholar
- Brunetti CR, Selegue JE, Monteiro A, French V, Brakefield PM, Carroll SB. The generation and diversification of butterfly eyespot color patterns. Curr Biol. 2001;11:1578–85.View ArticlePubMedGoogle Scholar
- Werner T, Koshikawa S, Williams TM, Carroll SB. Generation of a novel wing colour pattern by the Wingless morphogen. Nature. 2010;464:1143–8.View ArticlePubMedGoogle Scholar
- Martin A, Papa R, Nadeau NJ, Hill RI, Counterman BA, Halder G, Jiggins CD, Kronforst MR, Long AD, McMillan WO, Reed RD: Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand. Proc Natl Acad Sci U S A. 2012;109:12632–7.Google Scholar
- Keys DN, Lewis DL, Selegue JE, Pearson BJ, Goodrich LV, Johnson RL, Gates J, Scott MP, Carroll SB. Recruitment of a hedgehog regulatory circuit in butterfly eyespot evolution. Science. 1999;283:532–4.Google Scholar
- Saenko SV, French V, Brakefield PM, Beldade P. Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings. Philos Trans R Soc Lond B Biol Sci. 2008;363:1549–55.View ArticlePubMedPubMed CentralGoogle Scholar
- ffrench-Constant RH. Butterfly wing colours are driven by the evolution of developmental heterochrony. Butterfly wing colours and patterning by numbers. Heredity. 2012;108:592–3.View ArticlePubMedPubMed CentralGoogle Scholar
- Mcmillan WO, Monteiro A, Kapan DD. Development and evolution on the wing. Trends Ecol Evol. 2002;17:125–33.View ArticleGoogle Scholar
- Brisson JA, Ishikawa A, Miura T. Wing development genes of the pea aphid and differential gene expression between winged and unwinged morphs. Insect Mol Biol. 2010;19 Suppl 2:63–73.View ArticlePubMedGoogle Scholar
- Abouheif E, Wray GA. Evolution of the gene network underlying wing polyphenism in ants. Science. 2002;297:249–52.View ArticlePubMedGoogle Scholar
- Mann RS, Carroll SB. Molecular mechanisms of selector gene function and evolution. Curr Opin Genet Dev. 2002;12:592–600.View ArticlePubMedGoogle Scholar
- Carroll SB, Grenier JK, Weatherbee SD. From DNA to diversity: molecular genetics and the evolution of animal design. 2nd ed. Oxford: Wiley-Blackwell; 2004.Google Scholar
- Wolpert L. Cell boundaries: knowing who to mix with and what to shout or whisper. Development. 2003;130:4497–500.View ArticlePubMedGoogle Scholar
- Krupp JJ, Yaich LE, Wessells RJ, Bodmer R. Identification of genetic loci that interact with cut during Drosophila wing-margin development. Genetics. 2005;170:1775–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Weatherbee SD, Nijhout HF, Grunert LW, Halder G, Galant R, Selegue J, Carroll S. Ultrabithorax function in butterfly wings and the evolution of insect wing patterns. Curr Biol. 1999;9:109–15.View ArticlePubMedGoogle Scholar
- Halder G, Carroll SB. Binding of the Vestigial co-factor switches the DNA-target selectivity of the Scalloped selector protein. Development. 2001;128:3295–305.PubMedGoogle Scholar
- Martin A, Reed RD. Wingless and aristaless2 define a developmental ground plan for moth and butterfly wing pattern evolution. Mol Biol Evol. 2010;27:2864–78.View ArticlePubMedGoogle Scholar
- Oliver JC, Tong X-L, Gall LF, Piel WH, Monteiro A. A single origin for nymphalid butterfly eyespots followed by widespread loss of associated gene expression. PLoS Genet. 2012;8, e1002893.View ArticlePubMedPubMed CentralGoogle Scholar
- Monteiro A, Podlaha O. Wings, horns, and butterfly eyespots: how do complex traits evolve? PLoS Biol. 2009;7, e37.View ArticlePubMedGoogle Scholar
- Monteiro A. Gene regulatory networks reused to build novel traits: co-option of an eye-related gene regulatory network in eye-like organs and red wing patches on insect wings is suggested by optix expression. Bioessays. 2012;34:181–6.View ArticlePubMedGoogle Scholar
- Monteiro A, Glaser G, Stockslager S, Glansdorp N, Ramos D: Comparative insights into questions of lepidopteran wing pattern homology. BMC Developmental Biology. 2006;13:1–13.Google Scholar
- Ferguson LC, Jiggins CD. Shared and divergent expression domains on mimetic Heliconius wings. Evol Dev. 2009;11:498–512.View ArticlePubMedGoogle Scholar
- Ferguson LC, Maroja L, Jiggins CD. Convergent, modular expression of ebony and tan in the mimetic wing patterns of Heliconius butterflies. Dev Genes Evol. 2011;221:297–308.View ArticlePubMedGoogle Scholar
- Reed RD, Nagy LM. Evolutionary redeployment of a biosynthetic module: expression of eye pigment genes vermilion, cinnabar, and white in butterfly wing development. Evol Dev. 2005;7:301–11.View ArticlePubMedGoogle Scholar
- Koch PB, Lorenz U, Brakefield PM, ffrench-Constant RH. Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes. Dev Genes Evol. 2000;210:536–44.View ArticlePubMedGoogle Scholar
- Hines HM, Papa R, Ruiz M, Papanicolaou A, Wang C, Nijhout HF, McMillan WO, Reed RD. Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation. BMC Genomics. 2012;13:288.Google Scholar
- Stoehr AM, Walker JF, Monteiro A. Spalt expression and the development of melanic color patterns in pierid butterflies. Evodevo. 2013;4:6.View ArticlePubMedPubMed CentralGoogle Scholar
- Kronforst MR, Barsh GS, Kopp A, Mallet J, Monteiro A, Mullen SP, Protas M, Rosenblum EB, Schneider CJ, Hoekstra HE. Unraveling the thread of nature's tapestry: the genetics of diversity and convergence in animal pigmentation. Pigment Cell and Melanoma Research 2012;25(4):411–33.Google Scholar
- Gibert J-M, Peronnet F, Schlötterer C. Phenotypic plasticity in Drosophila pigmentation caused by temperature sensitivity of a chromatin regulator network. PLoS Genet. 2007;3, e30.View ArticlePubMedPubMed CentralGoogle Scholar
- Ekblom R, Galindo J. Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity. 2011;107:1–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Ferguson L, Lee SF, Chamberlain N, Nadeau N, Joron M, Baxter S, Wilkinson P, Papanicolaou A, Kumar S, Kee T-J, Clark R, Davidson C, Glithero R, Beasley H, Vogel H, Ffrench-Constant R, Jiggins C. Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locus. Mol Ecol. 2010;19 Suppl 1:240–54.View ArticlePubMedGoogle Scholar
- Carroll SB, Gates J, Keys DN, Paddock SW, Panganiban GE, Selegue JE, Williams JA. Pattern formation and eyespot determination in butterfly wings. Science. 1994;265:109–14.View ArticlePubMedGoogle Scholar
- Supple MA, Hines HM, Dasmahapatra KK, Lewis JJ, Nielsen DM, Lavoie C, RayDA, Salazar C, McMillan WO, Counterman BA. Genomic architecture of adaptive color pattern divergence and convergence in Heliconius butterflies. Genome Res. 2013;23:1248–57.Google Scholar
- Beldade P, Rudd S, Gruber JD, Long AD. A wing expressed sequence tag resource for Bicyclus anynana butterflies, an evo-devo model. BMC Genomics. 2006;7:130.View ArticlePubMedPubMed CentralGoogle Scholar
- Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11:R25.View ArticlePubMedPubMed CentralGoogle Scholar
- Conesa A, Götz S. Blast2GO: A Comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics. 2008;2008:1–12.View ArticleGoogle Scholar
- Daniels EV, Murad R, Mortazavi A, Reed RD. Extensive transcriptional response associated with seasonal plasticity of butterfly wing patterns. Mol Ecol. 2014;23:6123–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhan S, Merlin C, Boore JL, Reppert SM. The monarch butterfly genome yields insights into long-distance migration. Cell. 2011;147:1171–85.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang Y, Niu B, Gao Y, Fu L, Li W. CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics. 2010;26:680–2.View ArticlePubMedPubMed CentralGoogle Scholar
- The International Silkworm Genome Consortium. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol. 2008;38:1036–45.View ArticleGoogle Scholar
- Macias-Muñoz A, Smith G, Monteiro A, Briscoe AD. Transcriptome-wide differential gene expression in Bicyclus anynana butterflies: female vision-related genes are more plastic. Mol Biol Evol. 2016;33:79–92.View ArticlePubMedGoogle Scholar
- Rhen T, Metzger K, Schroeder A, Woodward R. Expression of putative sex-determining genes during the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina. Sex Dev. 2007;1:255–70.View ArticlePubMedGoogle Scholar
- Reed RD, Papa R, Martin A, Hines HM, Counterman BA, Pardo-Diaz C, Jiggins CD, Chamberlain NL, Kronforst MR, Chen R, Halder G, Nijhout HF, McMillan WO. Optix drives the repeated convergent evolution of butterfly wing pattern mimicry. Science. 2011;333:1137–41.View ArticlePubMedGoogle Scholar
- Levine B, Jean-Francois M, Bernardi F, Gargiulo G, Dobens L. Notch signaling links interactions between the C/EBP homolog slow border cells and the GILZ homolog bunched during cell migration. Dev Biol. 2007;305:217–31.View ArticlePubMedGoogle Scholar
- Sun J, Deng WM. Hindsight mediates the role of notch in suppressing hedgehog signaling and cell proliferation. Dev Cell. 2007;12:431–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Monteiro A, Chen B, Ramos DM, Oliver JC, Tong X, Guo M, Wang W-K, Fazzino L, Kamal F. Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies. J Exp Zool B Mol Dev Evol. 2013;320:321–31.View ArticlePubMedGoogle Scholar
- Ou J, Deng H-M, Zheng S-C, Huang L-H, Feng Q-L, Liu L. Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis. BMC Genomics. 2014;15:820.View ArticlePubMedPubMed CentralGoogle Scholar
- Binggeli O, Neyen C, Poidevin M, Lemaitre B. Prophenoloxidase activation is required for survival to microbial infections in Drosophila. PLoS Pathog. 2014;10, e1004067.View ArticlePubMedPubMed CentralGoogle Scholar
- Wittkopp PJ, Carroll SB, Kopp A. Evolution in black and white: genetic control of pigment patterns in Drosophila. Trends Genet. 2003;19:495–504.View ArticlePubMedGoogle Scholar
- Liu J, Lemonds TR, Popadić A. The genetic control of aposematic black pigmentation in hemimetabolous insects: insights from Oncopeltus fasciatus. Evol Dev. 2014;16:270–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Riedel F, Vorkel D, Eaton S. Megalin-dependent yellow endocytosis restricts melanization in the Drosophila cuticle. Development. 2011;138:149–58.View ArticlePubMedGoogle Scholar
- Zhan S, Guo Q, Li M, Li M, Li J, Miao X, Huang Y: Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation. Development. 2010;137:4083–90.Google Scholar
- Walter MF, Black BC, Afshar G, Kermabon A-Y, Wright TRF, Biessmann H. Temporal and spatial expression of the yellow gene in correlation with cuticle formation and DOPA decarboxylase activity in drosophila development. Dev Biol. 1991;147:32–45.Google Scholar
- Arakane Y, Dittmer NT, Tomoyasu Y, Kramer KJ, Muthukrishnan S, Beeman RW, Kanost MR: Identification, mRNA expression and functional analysis of several yellow family genes in Tribolium castaneum. Insect Biochem Mol Biol. 2010;40:259–66.Google Scholar
- Lloyd V, Ramaswami M, Krämer H. Not just pretty eyes: Drosophila eye-colour mutations and lysosomal delivery. Trends Cell Biol. 1998;8(7):257–9.Google Scholar
- Ma J, Plesken H, Treisman JE, Edelman-Novemsky I, Ren M. Lightoid and Claret: a rab GTPase and its putative guanine nucleotide exchange factor in biogenesis of Drosophila eye pigment granules. Proc Natl Acad Sci U S A. 2004;101:11652–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Reaume AG, Knecht DA, Chovnick A. The rosy locus in Drosophila melanogaster: xanthine dehydrogenase and eye pigments. Genetics. 1991;129:1099–109.PubMedPubMed CentralGoogle Scholar
- Wang Q, Zhao C, Bai L, Deng X, Wu C. Reduction of drosopterin content caused by a 45-nt insertion in Henna pre-mRNA of Drosophila melanogaster. Sci China Ser C Life Sci. 2008;51:702–10.View ArticleGoogle Scholar
- Wijnen B, Leertouwer HL, Stavenga DG. Colors and pterin pigmentation of pierid butterfly wings. J Insect Physiol. 2007;53:1206–17.View ArticlePubMedGoogle Scholar
- Stavenga DG, Stowe S, Siebke K, Zeil J, Arikawa K. Butterfly wing colours: scale beads make white pierid wings brighter. Proc Biol Sci. 2004;271:1577–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Shamim G, Ranjan SK, Pandey DM, Ramani R. Biochemistry and biosynthesis of insect pigments. Eur J Entomol. 2014;111:149–64.Google Scholar
- Nishikawa H, Iga M, Yamaguchi J, Saito K, Kataoka H, Suzuki Y,Sugano S, Fujiwara H: Molecular basis of wing coloration in a Batesian mimic butterfly, Papilio polytes. Sci Rep. 2013;3:3184.Google Scholar
- Richardson G, Ding H, Rocheleau T, Mayhew G, Reddy E, Christensen BM, Li J, Hall E, Tech V: An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes. Mol Biol. 2010;37:3199–205.Google Scholar
- Phillips AM, Smart R, Strauss R, Brembs B, Kelly LE. The drosophila black enigma: the molecular and behavioural characterization of the mutant allele. Gene. 2005;351:131–42.View ArticlePubMedGoogle Scholar
- Xia A-H, Zhou Q-X, Yu L-L, Li W-G, Yi Y-Z, Zhang Y-Z, Zhang Z-F: Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori. BMC Genomics. 2006;7:195.Google Scholar
- Ferguson LC, Green J, Surridge A, Jiggins CD. Evolution of the insect yellow gene family. Mol Biol Evol. 2011;28:257–72.Google Scholar
- Iwata M, Ohno Y, Otaki JM. Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue. PLoS One. 2014;9, e89500.View ArticlePubMedPubMed CentralGoogle Scholar
- Fu W, Duan H, Frei E, Noll M. Shaven and sparkling are mutations in separate enhancers of the Drosophila Pax2 homolog. Development. 1998;125:2943–50.PubMedGoogle Scholar
- Gibert JM, Marcellini S, David JR, Schlotterer C, Simpson P. A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor poils-au-dos, a repressor of achaete-scute. Dev Biol. 2005;288:194–205.View ArticlePubMedGoogle Scholar
- Galant R, Skeath JB, Paddock S, Lewis DL, Carroll SB. Expression pattern of a butterfly achaete-scute homolog reveals the homology of butterfly wing scales and insect sensory bristles. Curr Biol. 1998;8:807–13.View ArticlePubMedGoogle Scholar
- Chanut-Delalande H, Fernandes I, Roch F, Payre F, Plaza S. Shavenbaby couples patterning to epidermal cell shape control. PLoS Biol. 2006;4:e290.View ArticlePubMedPubMed CentralGoogle Scholar
- French V, Brakefield PM. Pattern Formation : A focus on notch in butterfly eyespots new observations of early and dynamic expression. Curr Biol. 2004;14:663–5.View ArticleGoogle Scholar
- Reed RD, Serfas MS. Butterfly wing pattern evolution is associated with changes in a Notch/Distal-less temporal pattern formation process. Curr Biol. 2004;14(13):1159–66.View ArticlePubMedGoogle Scholar
- Ash DM, Hackney JF, Jean-Francois M, Burton NC, Dobens LL. A dominant negative allele of the Drosophila leucine zipper protein Bunched blocks bunched function during tissue patterning. Mech Dev. 2007;124:559–69.View ArticlePubMedGoogle Scholar
- Monteiro A, Tong X, Bear A, Liew SF, Bhardwaj S, Wasik BR, Dinwiddie A, Bastianelli C, Cheong WF, Wenk MR, Cao H, Prudic KL: Differential expression of ecdysone receptor leads to variation in phenotypic plasticity across serial homologs. PLoS Genet. 2015;11, e1005529.Google Scholar
- Koch PB, Merk R, Reinhardt R, Weber P. Localization of ecdysone receptor protein during colour pattern formation in wings of the butterfly Precis coenia (Lepidoptera: Nymphalidae) and co-expression with Distal-less protein. Dev Genes Evol. 2003;212:571–84.Google Scholar
- Hiruma K, Riddiford LM. The molecular mechanisms of cuticular melanization: The ecdysone cascade leading to dopa decarboxylase expression in Manduca sexta. Insect Biochem Mol Biol. 2009;39:245–53.View ArticlePubMedGoogle Scholar
- Cherbas L, Lee K, Cherbas P. Identification of ecdysone response elements by analysis of the Drosophila Eip28/29 gene. Genes Dev. 1991;5:120–31.View ArticlePubMedGoogle Scholar
- Schubiger M. Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing. Development. 2005;132:5239–48.View ArticlePubMedGoogle Scholar
- Jindra M, Malone F, Hiruma K, Riddiford LM. Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm. Manduca sexta. 1996;272:258–72.Google Scholar
- Futahashi R, Fujiwara H. Regulation of 20-hydroxyecdysone on the larval pigmentation and the expression of melanin synthesis enzymes and yellow gene of the swallowtail butterfly, Papilio xuthus. Insect Biochem Mol Biol. 2007;37:855–64.View ArticlePubMedGoogle Scholar
- Chen L, Reece C, O’Keefe SL, Hawryluk GWL, Engstrom MM, Hodgetts RB. Induction of the early-late Ddc gene during Drosophila metamorphosis by the ecdysone receptor. Mech Dev. 2002;114:95–107.View ArticlePubMedGoogle Scholar