DOI: 10.5061/dryad.pnvx0k6jp
title: Data from: Testosterone regulates CYP2J19-linked carotenoid signal expression in male red-backed fairywrens (Malurus melanocephalus)

description: Carotenoid pigments produce most red, orange, and yellow colours in
 vertebrates. This coloration can serve as an honest signal of quality that
 mediates social and mating interactions, but our understanding of the
 underlying mechanisms that control carotenoid signal production, including
 how different physiological pathways interact to shape and maintain these
 signals, remains incomplete. We investigated the role of testosterone in
 mediating gene expression associated with a red plumage sexual signal in
 red-backed fairywrens (Malurus melanocephalus). In this species, males
 within a single population can flexibly produce either red/black nuptial
 plumage or female-like brown plumage. Combining correlational analyses
 with a field-based testosterone implant experiment and qPCR, we show that
 testosterone mediates expression of carotenoid-based plumage in part by
 regulating expression of CYP2J19, a ketolase gene associated with
 ketocarotenoid metabolism and pigmentation in birds. This is the first
 time that hormonal regulation of a specific genetic locus has been linked
 to carotenoid production in a natural context, revealing how endocrine
 mechanisms produce sexual signals that shape reproductive success.
description: (a) Plasma sample collection and quantifying circulating
 carotenoids             We collected samples and
 conducted experiments (below) on free-living red-backed fairywrens
 captured in mist nets at our long-term study site in Samsonvale, QLD,
 Australia (27°27’ S, 152°85’ E). We collected blood samples (20–70 µl)
 from the wing vein using heparinized microcapillary tubes from May -
 August 2017 and 2018 during the non-breeding season, a period when most
 males were actively moulting into their breeding season plumage. Blood was
 centrifuged for 5 min at 10,000 rpm, after which the plasma was separated
 from the packed cells and stored at -20° C until transport to the US where
 samples were stored at -80° C until high performance liquid chromatography
 (HPLC) analysis.             Plumage score was recorded
 at time of capture following prior methods (Karubian, 2002), yielding
 total ornamentation scores that ranged from 0 (brown) to 100 (red/black).
 Based on this score, males were labelled as either “unornamented” (brown
 plumage, score &lt;33), “intermediate” (mixed plumage, score between
 33 and 66), or “ornamented” (red/black plumage, score &gt;66). Females
 always have completely brown plumage (plumage score = 0) and are therefore
 considered unornamented. Timing of moult into ornamented plumage is
 variable (Welklin et al. unpub. data), similar to other recorded
 <i>Malurus</i> species (Mulder & Magrath, 1994),
 meaning that male plumage at time of capture and sample collection may
 differ from the “final” plumage colour score the male expressed later in
 the breeding season. Males can breed in unornamented or ornamented 
 plumage or serve as auxiliaries with unornamented plumage (helpers) at the
 nest (Webster, Karubian, & Schwabl, 2010). Because we were
 interested in differences between unornamented and ornamented plumage, we
 documented the “final” plumage score of colour-banded individuals on
 November 1<sup>st</sup> (the approximate mid-point of the
 breeding season) and used this “final” score for analyses of unornamented
 males, ornamented males, and females; we excluded from analysis the
 relatively small sub-set of birds whose “final” plumage score was
 intermediate (n = 11). We assigned either minimum or known age (age range
 1–7 years, 77%, 123 of 160, were of known age) to all birds at the time of
 sample collection using nestling banding records or extent of skull
 ossification (ossification scale modified from (Pyle, Howell, Yunick,
 & Desante, 1987), and we have validated this scale within this
 species multiple times). Qualitatively similar results were obtained in
 analyses run with these age criteria, or using only known-age birds (see
 electronic supplementary material, Table S1).
             We used high performance liquid chromatography (HPLC)
 to identify and quantify the concentration of carotenoids in the plasma,
 following the methods of Rowe and McGraw 2008 (Rowe & McGraw,
 2008). We analysed carotenoids in 160 plasma samples (n=42 females, n=29
 unornamented males, n=89 ornamented males).  To assess the relationship
 between circulating ketocarotenoid levels and plumage phenotype, we ran a
 linear mixed effect model with the lme function in the R package nlme
 (Pinheiro, Bates, DebRoy, Sarkar, & R Core Team, 2018), R v. 3.6.0
 (R Core Team, 2019). The model included total circulating ketocarotenoid
 concentration (i.e. the sum of alpha-doradexanthin, astaxanthin,
 adonirubin, and canthaxanthin concentrations) as the response variable and
 the following predictor variables: (1) phenotype (female vs. unornamented
 male vs. ornamented male); (2) age (as a continuous variable); (3) year of
 sample collection; and (4) the interaction between phenotype and age. To
 control for repeated measures of the same individual across years, we
 added individual as a random effect (n=13 individuals sampled both years).
 Residuals were inspected visually for homoscedasticity, and we used the
 varIdent function to control for heterogeneity of variance between groups.
 Year of sample collection did not improve model fit (i.e, it did not
 improve AIC by more than 2 and the p-value of the Year variable was
 greater than 0.05) and was therefore dropped. We tested the model for
 significance of phenotype with a Tukey’s posthoc test using the glht
 function in the R package multcomp (Hothorn, Bretz, & Westfall,
 2008).    (b) Testosterone implantation and liver
 sample collection             We collected liver
 samples from breeding, but not auxiliary helper, red-backed fairywrens in
 November 2017, to control for potentially confounding underlying
 differences in endocrine or genetic profiles that may exist between
 auxiliary non-breeding vs. breeding individuals (Lindsay, Webster, Varian,
 & Schwabl, 2009). First, three breeding unornamented males were
 implanted with testosterone. At time of initial capture and implantation,
 around 10 feathers were plucked from the centre of the back to induce
 feather replacement at that location. Implants were composed of beeswax
 (73% by weight; Sigma-Aldrich, St. Louis, MO) and hardened frozen peanut
 oil (24% by weight; ACROS Organics, NJ, USA) that were mixed in a water
 bath at 67° C. Once the beeswax/peanut oil mixture was melted, crystalline
 testosterone (3% by weight; Sigma-Aldrich, St. Louis, MO, USA) was
 dissolved in 2.5 μl of 200 proof
 ethanol (Fisher Bioreagants<sup>TM</sup>). The implants were
 formed by feeding partially solidified wax through the tip of a syringe,
 resulting in implants of 2 x3.2mm weighing between 19.8 and 20.7 mg.
 Testosterone concentration in the beeswax carrier was scaled to produce
 high physiological concentrations found in circulation during the breeding
 season (Lindsay et al., 2009). Implants were inserted subcutaneously using
 forceps above the thigh into a small (2-3 mm) skin incision that was
 sealed with veterinary skin adhesive. After confirming that the incision
 was completely sealed and the bird was in good condition, the bird was
 released. Three unornamented males were implanted with sham controls
 (beeswax/peanut oil implant with no testosterone) and also had around 10
 back feathers plucked.             Implanted birds were
 recaptured 10-12 days post-implantation for liver sample collection, a
 time period that allowed for growth of pin feathers in the plucked plumage
 patches (red pins in testosterone-implanted males, and a mix of red and
 brown pins in the sham-implanted males, consistent with what had been
 observed in another feather-plucking study in this species (Karubian,
 Lindsay, Schwabl, & Webster, 2011)). We were unable to recapture
 one of the sham-implanted birds after implantation. Instead, we captured
 and obtained samples from one additional breeding unornamented male, who
 was not implanted, to include in our control group. Following re-capture
 on territories in mist nets, birds were immediately sacrificed by cervical
 dislocation. Body dissection was performed in the field, and the right
 lower lobe of the liver was removed and stored in 1mL of RNAlater storage
 buffer (ThermoFisher Scientific), and immediately placed on dry ice.
 Samples were stored at -80° C until RNA extraction. In addition to the
 three testosterone-implanted unornamented males and the three control
 unornamented males (two with sham implants, one without an implant), we
 also collected liver samples for three ornamented breeding males and three
 breeding females (without implants). All birds sacrificed were seen paired
 with a male or female within two weeks prior to sample collection, and all
 samples were collected within a period of 9 days. Circulating androgens
 were measured using an established radioimmunoassay protocol for this
 species (full methods in (Barron, Webster, & Schwabl, 2015;
 Lindsay et al., 2009)); the intra-assay coefficient of variation was
 8.68%. Testosterone-implanted birds were confirmed to have high
 concentrations of circulating androgens at time of collection (mean=3027
 pg/ml, range=2198-4065 pg/ml), which is within the natural range of
 androgens for breeding ornamented males in this species (Lindsay et al.,
 2009). Ornamented males also had similarly high levels of androgens at
 time of collection (mean=1834 pg/ml, range=1124-2925 pg/ml). We were
 unable to obtain samples to assay testosterone concentrations for control
 unornamented males or females. (c) Quantifying relative
 expression of CYP2J19             To extract messenger
 RNA (mRNA), we removed liver tissue from the RNAlater buffer and
 homogenized it in a Qiagen TissueRuptor. We used a Qiagen RNAeasy
 mini-kit, following manufacturer’s instructions, and reverse transcribed
 the mRNA to cDNA with a Superscript IV first strand synthesis kit
 (Invitrogen). All qPCR reactions were run on CFX96 Touch™ Real-Time
 PCR Detection System (BioRad) with CFX Maestro Software (BioRad), using
 PowerUp SYBR Green Master Mix (Thermofisher Scientific). For measurements
 of CYP2J19 expression, we used qPCR primers CYP2J2-2F and CYP2J2-2R (Mundy
 et al., 2016). We assayed gene expression in triplicate for each sample
 and normalized the data using the housekeeping gene GAPDH, using primers
 Gg_GAPDH_qPCR_F and Gg_GAPDH_qPCR_R (Lopes et al., 2016). Reaction
 conditions for qPCR were tested and optimized using a standard curve
 produced by creating a serial dilution of a pool of all cDNA samples.
 Efficiencies ranged from 95%-105%, and we analysed qPCR data using the
 delta-delta Ct method (Livak & Schmittgen, 2001), further
 described in electronic supplementary material, Methods.
             We confirmed that there was no effect of presence or
 absence of the sham implant on gene expression within unornamented males
 (see electronic supplementary material, Methods), and homoscedasticity was
 confirmed with a Breusch Pagan test (p&gt;0.05). We tested for
 statistical differences in liver CYP2J19 expression (log fold change)
 between phenotypes with an ANOVA, using the aov function in R, followed by
 a Tukey’s posthoc test using the TukeyHSD function in R.
 <b>Supplementary Methods:</b>
 Analyzing qPCR data with delta-delta CT
 method We analysed qPCR data using the
 delta-delta Ct method [S1], which reports gene expression (mRNA abundance)
 for the gene of interest as the fold change in expression
 (2<sup>-</sup><sup>DDCt</sup>), normalized to a
 housekeeping gene and calibrated to a “calibrator sample”. We use GAPDH as
 our housekeeping gene, as it is often used as a housekeeping gene for qPCR
 analysis in birds [S2–S5]. We averaged the DCt (Ct CYP2J19 – Ct GAPDH) for
 all three females and used that average as our calibrator sample. We
 present our results as log fold change. Using the average female DCt value
 as our calibrator sets the average log fold change value for females at
 zero, allowing for easier visualization of the difference in relative gene
 expression between phenotypes. We also ran this analysis with only one
 female as our calibrator sample, and our statistical results remain
 exactly the same since these are all relative expression levels.
 Assessing the effect of the presence of absence of a
 sham implant In order to assess whether or
 not having a sham implant affected expression levels for those
 unornamented males, we combined the two sham-implanted males and the
 unmanipulated unornamented male into one phenotype category we called
 “control unornamented male.” We ran a linear model using the lm function
 in R with CYP2J19 expression (log fold change) as the response variable,
 and presence of an implant (yes vs. no) nested within phenotype (female
 vs. control unornamented male vs. testosterone-implanted unornamented male
 vs. ornamented male) as the response variable. We found no interaction
 between phenotype and implant, and the implant variable did not improve
 the fit from the model, suggesting there was no significant effect of not
 having the sham implant within our control unornamented males. In
 addition, we evaluated raw data and confirmed that the log fold change for
 the unmanipulated male (2.20) was similar to that of the two sham implant
 males (1.95 and 2.19, and can be seen in figure S2), and the variance of
 the log fold change for control unornamented males (all three males) was
 very small, as can be seen in the standard error bars in Figure 2. Taken
 all together, we therefore dropped the implant variable from our
 model.    
description: The first data set “Khalil_etal_2020_HPLC_data.csv”
 is the circulating carotenoid data from the High Performance Liquid
 Chromatography (HPLC) Analysis. This data was used for the circulating
 kecarotenoid model. The data
 set consists of (in this order): 
 Year of sample collection
 Sample number
 Day of sample collection 
 ABBBS number – metal band number, a unique
 identifier Color bands –
 color band combination, another unique identifier, and the one used in the
 model for the random effect
 Sex
 Age - those with a “+” were of minimum age, since
 they are considered at least that age.       Those without a “+” are of
 known age. Age_min – same
 information as age, but without the “+” to be used in analysis. When
 running the model with only known age birds (Table S1), we filtered out
 individuals that had a “+” in the Age column.
 The next six columns are the circulating carotenoids
 -ador (alpha-doradexanthin), asta (astaxanthin), lut (lutein), adoni
 (adonirubin), zea (zeaxanthin), and canth
 (canthaxanthin).
 Breeding_phenotype – the breeding phenotype (as of
 November 1<sup>st</sup>) used for the analysis. F is female,
 DM is unornamented male, and BM is ornamented male.
  
 The second data set “Khalil_etal_2020_qpcr_data.xlsx”
 is the qPCR ct data from the CYP2J19 and GAPDH expression analysis. This
 data was used to calculate log fold change using the delta-delta ct method
 described in text. The data
 set consists of three sheets:
 1) gapdh_ct – this is the ct data using the GAPDH
 primers. This includes 
 The well the sample was in
 The cq
 The sample number
 The phenotype of the sample. F is female, T-DM is
 testosterone-implanted unornamented male, DM is control unornamented male,
 and BM is ornamented male.
 The average GAPDH ct, calculated from the cq column
 as each sample was run in triplicate 
 2) cyp2j19_ct – this is the ct data using the CYP2J19
 primers. This includes the same type of information as the gapdh_ct
 sheet 3) delta_delta_ct_math –
 this is all the math to calculate the log fold change using the delta
 delta ct method. This includes: 
 The sample number
 The phenotype
 If the control unornamented male (DM) had a sham
 implant or no implant The
 average GAPDH ct from sheet 1
 The average CYP2J19 cqtfrom sheet
 2 The delta ct (CYP2J19 ct –
 GAPDH ct) The average delta
 ct for females. This is used as our “calibrator” sample, as described in
 the supplementary methods
 Delta delta ct (delta ct – average female delta
 ct) Fold change (2^delta
 delta ct) Log fold change.
 This value is used in the ANOVA and in figures. 
 4) Intra-assay CV - this is the math for how
 intra-assay coefficient of variation for the GAPDH and CYP2J19 qPCR
 assays