Testosterone regulates CYP2J19-linked carotenoid signal expression in male red-backed fairywrens (Malurus melanocephalus)

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. Methods (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 <33), “intermediate” (mixed plumage, score between 33 and 66), or “ornamented” (red/black plumage, score >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 Malurus 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 1st (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 BioreagantsTM). 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>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. Supplementary Methods: 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-DDCt), 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.类胡萝卜素（carotenoid）色素是脊椎动物体内绝大多数红色、橙色及黄色体色的成因。此类体色可作为反映个体品质的诚实信号，介导社会互动与配偶选择行为，但目前学界对调控类胡萝卜素信号生成的底层机制——包括不同生理通路如何相互作用以塑造并维持此类信号——的认知仍不完整。本研究以红背细尾鹩莺（*Malurus melanocephalus*）为对象，探究睾酮（testosterone）介导与红色羽饰性信号相关基因表达的作用机制。该物种种群内的雄性可灵活表达两种羽型：一是红黑色的婚羽，二是类似雌性的棕羽。本研究结合相关性分析、野外睾酮植入实验与实时荧光定量PCR（qPCR）技术，证实睾酮可通过调控鸟类体内与类胡萝卜素酮衍生物代谢及色素沉着相关的酮酶基因CYP2J19的表达，部分介导基于类胡萝卜素的羽饰表达。本研究首次在自然情境下将特定基因位点的激素调控与类胡萝卜素生成建立关联，揭示了内分泌机制如何通过生成性信号影响繁殖成功率。 方法 (a) 血浆样本采集与循环类胡萝卜素定量 本研究于澳大利亚昆士兰州萨姆森瓦尔（Samsonvale，27°27′ S，152°85′ E）的长期研究样地中，通过雾网捕获野生红背细尾鹩莺，并开展样本采集与下述实验。2017年与2018年的5月至8月（非繁殖季），我们使用肝素化毛细管从翼静脉采集20~70 µl血液样本——此时多数雄性正处于向繁殖季羽型换羽的阶段。血液经10000 rpm离心5分钟后，分离血浆与压紧的血细胞，将血浆置于-20℃保存，待转运至美国后，再于-80℃保存至高效液相色谱（HPLC）分析阶段。 捕获时参照已发表方法（Karubian, 2002）记录羽饰评分，总装饰性评分范围为0（棕羽）至100（红黑羽）。依据该评分，雄性可分为三类：无装饰型（棕羽，评分<33）、中间型（混合羽色，评分33~66）与装饰型（红黑羽，评分>66）。雌性始终为通体棕羽（羽饰评分=0），因此归类为无装饰型。雄性换羽形成装饰羽的时间存在个体差异（Welklin等，未发表数据），这与已报道的其他细尾鹩莺属（*Malurus*）物种一致（Mulder & Magrath, 1994），即捕获采样时的雄性羽色可能与其繁殖季后期呈现的“最终”羽色评分存在差异。雄性可在无装饰羽或装饰羽状态下繁殖，也可作为辅助者（育雏帮手）以无装饰羽状态参与巢内活动（Webster, Karubian, & Schwabl, 2010）。鉴于本研究关注无装饰羽与装饰羽个体间的差异，我们于11月1日（繁殖季大致中点）对佩戴彩色脚环的个体记录其“最终”羽饰评分，并以该评分作为无装饰雄性、装饰雄性与雌性的分析依据；最终将11只评分为中间型的个体排除在分析之外。采样时，我们通过雏鸟环志记录或头骨骨化程度（骨化评分改编自Pyle等, 1987）为所有个体确定最小年龄或已知年龄（年龄范围1~7岁，160只个体中有123只为已知年龄，占比77%）；本研究团队已多次在该物种中验证该评分体系的有效性。无论采用上述年龄判定标准，还是仅使用已知年龄个体进行分析，均得到了定性一致的结果（详见电子补充材料表S1）。 参照Rowe与McGraw（2008）的方法，我们采用高效液相色谱（HPLC）对血浆中的类胡萝卜素进行定性与定量分析。本研究共分析了160份血浆样本，其中雌性42份、无装饰雄性29份、装饰雄性89份。为探究循环类胡萝卜素酮衍生物水平与羽饰表型间的关联，我们使用R软件nlme包中的lme函数构建线性混合效应模型（R版本3.6.0，R Core Team, 2019；Pinheiro等, 2018）。该模型以循环类胡萝卜素酮衍生物总浓度（即α- doradexanthin、虾青素、adonirubin与斑蝥黄浓度之和）为响应变量，纳入以下预测变量：（1）表型（雌性、无装饰雄性、装饰雄性）；（2）年龄（连续变量）；（3）采样年份；（4）表型与年龄的交互项。为控制同一个体跨年度重复采样带来的偏差，我们将个体身份作为随机效应纳入模型（共13只个体于两年间均被采样）。通过目视检查残差的同方差性，并使用varIdent函数控制组间方差异质性。采样年份未提升模型拟合度（即赤池信息准则AIC改善量未超过2，且年份变量的p值>0.05），因此将其从模型中移除。我们使用R软件multcomp包中的glht函数，通过Tukey事后检验对模型中表型变量的显著性进行验证（Hothorn等, 2008）。 (b) 睾酮植入与肝脏样本采集 2017年11月，我们仅从繁殖个体而非辅助育雏者的红背细尾鹩莺体内采集肝脏样本，以控制繁殖个体与非繁殖辅助个体间潜在的内分泌或基因表达差异带来的混淆因素（Lindsay等, 2009）。首先，我们为3只繁殖期无装饰雄性植入睾酮。初次捕获并植入时，我们从其背部中央拔下约10根羽毛，以诱导该区域羽毛再生。植入剂由蜂蜡（质量占比73%，Sigma-Aldrich，美国密苏里州圣路易斯）与硬化冷冻花生油（质量占比24%，ACROS Organics，美国新泽西州）在67℃水浴中混合制成。待蜂蜡与花生油混合物熔融后，将结晶睾酮（质量占比3%，Sigma-Aldrich，美国密苏里州圣路易斯）溶于2.5 μl 200度乙醇（Fisher Bioreagants™）中。通过注射器针头将半固化的蜡液挤出制成植入剂，最终得到尺寸为2×3.2mm、重量在19.8~20.7 mg之间的植入体。蜂蜡载体中的睾酮浓度经过校准，可使循环睾酮浓度达到繁殖季的生理高水平（Lindsay等, 2009）。使用镊子在大腿上方做2~3mm的皮肤切口，将植入剂皮下植入，随后用兽用皮肤粘合剂闭合切口。确认切口完全闭合且个体状态良好后，将其放归野外。另外3只无装饰雄性植入假对照剂（仅含蜂蜡与花生油，不含睾酮），同时也拔下背部约10根羽毛。 植入术后10~12天，我们对植入个体进行重捕以采集肝脏样本，此时拔毛区域已长出羽芽：睾酮植入雄性的羽芽为红色，假植入雄性的羽芽则为红棕混合色，这与该物种此前拔毛实验的观测结果一致（Karubian等, 2011）。但其中1只假植入个体未能被重捕，因此我们额外捕获1只未植入的繁殖期无装饰雄性，纳入对照组。在领域内通过雾网重捕后，我们立即通过颈椎脱臼法处死个体。野外解剖后，摘取肝脏右叶，将其置于1 mL RNAlater储存缓冲液（ThermoFisher Scientific）中，并立即放入干冰保存。样本于-80℃保存至RNA提取阶段。除3只睾酮植入无装饰雄性与3只对照无装饰雄性（2只假植入、1只未植入）外，我们还采集了3只繁殖期装饰雄性与3只繁殖期雌性（均未植入）的肝脏样本。所有处死个体均在采样前两周内被观测到有配偶，且所有样本的采集时间跨度不超过9天。采用该物种已建立的放射免疫测定法检测循环雄激素水平（详细方法见Barron等, 2015；Lindsay等, 2009），批内变异系数为8.68%。睾酮植入个体在采样时的循环雄激素浓度较高（平均值为3027 pg/ml，范围2198~4065 pg/ml），处于该物种繁殖期装饰雄性的天然雄激素浓度范围内（Lindsay等, 2009）。装饰雄性在采样时的雄激素水平同样较高（平均值为1834 pg/ml，范围1124~2925 pg/ml）。但我们未能采集到对照无装饰雄性与雌性的样本以检测其睾酮浓度。 (c) CYP2J19相对表达量定量 提取信使RNA（mRNA）时，我们先将肝脏组织从RNAlater缓冲液中取出，使用Qiagen TissueRuptor均质机进行匀浆。随后参照试剂盒说明书，使用Qiagen RNeasy Mini试剂盒提取RNA，并通过Superscript IV第一链合成试剂盒（Invitrogen）将mRNA反转录为互补DNA（cDNA）。所有实时荧光定量PCR（qPCR）反应均在CFX96 Touch™实时PCR检测系统（BioRad）上进行，配套使用CFX Maestro软件（BioRad）与PowerUp SYBR Green预混液（ThermoFisher Scientific）。检测CYP2J19的表达时，我们使用引物对CYP2J2-2F与CYP2J2-2R（Mundy等, 2016）。每个样本设置3次技术重复，并以内参基因GAPDH对数据进行标准化，内参引物为Gg_GAPDH_qPCR_F与Gg_GAPDH_qPCR_R（Lopes等, 2016）。通过对所有cDNA样本混合液进行梯度稀释制作标准曲线，对qPCR反应条件进行优化。扩增效率介于95%~105%之间，我们采用ΔΔCt法分析qPCR数据（Livak & Schmittgen, 2001），详细步骤见电子补充材料方法部分。 我们证实假植入与否对无装饰雄性的基因表达无显著影响（详见电子补充材料方法部分），且通过Breusch-Pagan检验确认了方差同质性（p>0.05）。我们使用R软件中的aov函数进行单因素方差分析（ANOVA），检验不同表型个体间肝脏CYP2J19表达量（对数折叠变化）的统计学差异，随后通过TukeyHSD函数进行Tukey事后检验。 补充方法 ΔΔCt法分析qPCR数据 我们采用ΔΔCt法分析qPCR数据[S1]，该方法以管家基因作为内参，并以“校准样本”作为基准，将目的基因的表达量（mRNA丰度）表示为表达折叠变化（2^-ΔΔCt）。本研究选用GAPDH作为管家基因，因其是鸟类qPCR分析中常用的内参基因[S2~S5]。我们将3只雌性的ΔCt值（ΔCt = Ct CYP2J19 – Ct GAPDH）取平均值，以此作为校准样本。本研究以对数折叠变化展示结果。以雌性平均ΔCt值作为校准样本，可使雌性的平均对数折叠变化值为0，便于直观比较不同表型间的相对基因表达差异。若仅使用1只雌性作为校准样本，分析结果也不会发生改变，因为所有数据均为相对表达量。 评估假植入与否的影响 为评估假植入对无装饰雄性基因表达水平的影响，我们将2只假植入雄性与1只未植入的无装饰雄性合并为“对照无装饰雄性”表型组。我们使用R软件中的lm函数构建线性模型，以CYP2J19表达量（对数折叠变化）为响应变量，将“是否植入（是/否）嵌套于表型（雌性、对照无装饰雄性、睾酮植入无装饰雄性、装饰雄性）”作为预测变量。分析结果显示，表型与植入与否之间无交互效应，且植入变量未提升模型拟合度，表明在对照无装饰雄性中，是否接受假植入对基因表达无显著影响。此外，我们对原始数据进行评估，证实未植入雄性的对数折叠变化值（2.20）与2只假植入雄性（1.95和2.19，详见图S2）相近；且3只对照无装饰雄性的对数折叠变化方差极小，如图2中的标准误棒所示。综合以上结果，我们最终将植入变量从模型中移除。