Evolutionary redeployment of a biosynthetic module: expression of eye pigment genes vermilion, cinna

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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

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Ommochrome synthesis in a flys eye
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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

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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.

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RNA Extration

• Pupal wings and larval imaginal discs were
  dissected
• RT-PCR used

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RNA Extraction (cont)

• RT-PCR explained
• (Reverse Transcriptase)

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Thin Layer Chromatography

• Method extracts and separates tryptophan-derived
  pigments from dried butterfly wings
• Sample spotted on silica gel
• Run in 31 phenolwater developing solvent
• Rf values were calculated
• Separated compounds were isolated and analyzed

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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

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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

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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

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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-AGTCCAGGCCGGTGGTNGG
  YTCRTC
• Produced several bands, one being right size,
  excised out and cloned

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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

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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

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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

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Results
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Identification of xanthommatin in red V. cardui
wing scales

• TLC analysis performed to determine which
  ommochromes were present in V. cardui wings
• Rf0.37, 450 nm peak consistent with xanthommatin
• Rf0.16, 450 nm peak suggests it might represent
  dihydro-xanthommatin
• Rf0.57 remains unknown and no similar compound
  was observed in Nijhouts (1997) work with
  another nymphalid

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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

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Melanin development preceds xanthommatin
development in V. cardui wings

• Pigment development begins a few days before
  emergence

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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

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Sequencing of V. cardui orthologs of actin,
vermilion, cinnabar, and, white
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Sequencing of V. cardui orthologs of actin,
vermilion, cinnabar, and, white
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Sequencing of V. cardui orthologs of actin,
vermilion, cinnabar, and, white
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High levels of vermilion, cinnabar, and white
transcription during scale development
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Spatially complex patterns of pigment gene
transcription
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Spatially complex patterns of pigment gene
transcription
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Discussion
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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

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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

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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

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Complex relationship between spatial pigment
patterns and gene expression

• Complex association between pigment gene
  transcription and adult pigment patterns
• Ommochrome gene synthesis doesnt show 11
  relationship with the time and place pigments
  synthesized
• All 3 genes were expressed in tissues where
  ommochromes are synthesized

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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