Evolutionary redeployment of a biosynthetic

module: expression of eye pigment genes

vermilion, cinnabar, and white in butterfly
wing development

By: Robert D. Reed and Lisa M. Nagy

Ommochrome synthesis in a fly’s eye
Materials and Methods
 Rearing and staging
 RNA extraction
 Thin layer chromatography
 Tryptophan incorporation assays
 Gene cloning and sequence analysis
 Quantitative real-time PCR
 In situ hybridization
Rearing and Staging
 Larvae fed artificial diet
 Larvae and pupae
maintained at 25
degrees Celsius
 Time-based staging
only considered
approximate because of
individual variation in
the relationship
between developmental
time and developmental
stage.
RNA Extration

 Pupal wings and

larval imaginal discs
were dissected
 RT-PCR used
RNA Extraction (cont)

 RT-PCR explained
(Reverse Transcriptase)
Thin Layer Chromatography
 Method extracts and
separates tryptophan-
derived pigments from
dried butterfly wings
 Sample spotted on silica
gel
 Run in 3:1 phenol:water
developing solvent
 Rf values were calculated
 Separated compounds
were isolated and analyzed
Tryptophan incorporation assays
 Pupae in late-stage were injected with
14C-labeled tryptophan

 Adults were frozen within 6 hours of

emergence
 Dorsal and ventral scales were peeled off the
wings
 Scales were exposed to autoradiographic film
for 9 days at -80o
Gene Cloning and Sequencing Analysis

 cDNA prepared using total RNA pooled

from fifth (last)-instar wing discs and
pupal wings
 Sequenced actin from randomly selected
clone
 Amplified vermilion using nested PCR
Gene cloning and sequence analysis

 Nested PCR for vermilion

 First amplification used primers:
 5’-GARYTNTGGTTYAARCARATH and
5’-NARNSWRTCDATRTCCAT
 No visible products on agarose gel
 Second amplification used primers:
 5’-CAYGAYGARCAYYTNTTYATH and
5’-RTCNARRAANACYTTRTA
 Desired size band excised out
Gene cloning and sequence analysis
 Amplified cinnabar using degenerate primers
 5’-ATGATGATHGCNYTNCCNAAYCARG and 5’-
RTARTTRTACATNGCNARRTC
 Cloned
 Two rounds of partially nested CODEHOP PCR used to amplify
white
 Round 1: used primers 5’-GCNMGNTGYGCNTAYGTNCARC and
5’-AGTCCAGGCCGGTGGTNGGYTCRTC
 Produced several bands, one being right size and excised out
 Round 2: used as a template for CODEHOP PCR with primers 5’-
GGTTCCGGCGCCGGNAARWSNAC and 5’-
AGTCCAGGCCGGTGGTNGGYTCRTC
 Produced several bands, one being right size, excised out and cloned
Gene cloning and sequence analysis

 Clones were sequenced

 Sequences with high degree of
similarity to D. melanogaster vermilion,
cinnabar, white, and actin were
translated and aligned from other taxa
Quantitative real-time PCR
• Locus specific primers used for
SYBR Green real-time PCR
• Relative transcription levels
were calculated using a
standard curve from a control
dilution series
• Vermilion, cinnabar, and white
data were normalized using
actin transcription levels
• RNA-only negative controls
were run for each experiment to
rule out genomic DNA
contamination
In situ hybridization
 Riboprobes were synthesized with dual-promoter verctors
bearing vermilion and cinnabar fragment inserts
 Larvae anesthetized in ice water, wing discs dissected
 Riboprobes were heat denatured and diluted
 Discs were placed in diluted buffer and incubated for 24 hours
 Discs were developed in the dark and monitored until
appropriate levels of colored precipitate formed, at which time
the reaction was stopped
Results
Identification of xanthommatin in red V.
cardui wing scales
 TLC analysis performed to determine which
ommochromes were present in V. cardui wings
 Rf=0.37, 450 nm peak consistent with xanthommatin
 Rf=0.16, 450 nm peak suggests it might represent
dihydro-xanthommatin
 Rf=0.57 remains unknown and no similar compound
was observed in Nijhout’s (1997) work with another
nymphalid
Identification of xanthommatin in red V.
cardui wing scales (cont)
• Tryptophan incorporation
in adult wing scales
• Dorsal surface, spatial
patterns of tryptophan
incorporation correlated
with only red pattern
elements
• Ventral surface,
tryptophan incoroporation
was seen in brown and tan
spots as well as some
black areas
Melanin development preceds xanthommatin
development in V. cardui wings
• Pigment
development begins
a few days before
emergence
Sequencing of V. cardui orthologs of
actin, vermilion, cinnabar, and, white
• Actin was identified
• 267 bp sequence
• 65 C-term amino acid
• Portion of the 3’ untranslated region of the
mRNA
• Amino acid similarity with other animals
was too high to allow for informative
phylogenetic analysis
Sequencing of V. cardui orthologs of
actin, vermilion, cinnabar, and, white
Sequencing of V. cardui orthologs of
actin, vermilion, cinnabar, and, white
Sequencing of V. cardui orthologs of
actin, vermilion, cinnabar, and, white
High levels of vermilion, cinnabar, and white
transcription during scale development
Spatially complex patterns of pigment
gene transcription
Spatially complex patterns of pigment
gene transcription
Discussion
Evolution of ommochrome synthesis
 Presence of ommochromes in insect eyes
appears to be nearly ubiquitous
 Visual filtering is the ancestral function of
ommochromes in insects
 Noneye contexts appear to be derived and
lineage specific
 Only known examples of ommochromes used as
wing scale pigments are from nymphalid butterflies
 Synthesis of ommochromes in butterfly wing scales
regarded as an evolutionary novelty
Unusual sequence of pigment synthesis
in V. cardui
 Proposed that ommochrome development
precedes melanin development in butterflies
 V. cardui shows an exception to this model
 Yellow pigment 3-OHK in Heliconius erato has
been observed to appear after melanin, only a few
hours before adult emergence
 Argues that modes of pigment development timing
in butterflies may not be as conserved as
previously thought
Gene expression in the context of
butterfly wing ommochrome synthesis
 Pupal wing discs in culture were found to
uptake tryptophan and synthesize
ommochromes in vitro
 Biochemical machinery needed to synthesize
ommochromes may be found within butterfly wings
 In this study, ommochrome gene expression
assays in wings occurred as early as the fifth
instar
 Major pigment peaks occurred 5-7 days before
pigments appeared
Complex relationship between spatial
pigment patterns and gene expression
 Complex association between pigment
gene transcription and adult pigment
patterns
 Ommochrome gene synthesis doesn’t
show 1:1 relationship with the time and
place pigments synthesized
 All
3 genes were expressed in tissues
where ommochromes are synthesized
A hypothetical model of ommochrome
synthesis in butterfly wings
 Model of ommochrome synthesis in D.
melanogaster may be extended to
butterfly wing scales
 Several features remain speculative
 Ommochromes are consistently associated
with granules in the eyes, integument, and
nervous systems of various other insects