Reproductive workers insufficiently signal their reproductive ability in a paper wasp

Colony census in the field Queens and nests attached under the eaves of buildings were collected in the field just before emergence of the first worker from the end of April to early June. The pedicels of each nest were vertically attached to the base of lidded transparent plastic cases (19 × 13 × 6 cm) with glue (Konishi G17). The individually marked queen (all colonies were single-foundress nests) was released on the original nest with weak immobilization on ice. After release, the case was fixed with metal wire within 15 cm of the original nest sites at midnight. No apparent queen loss was observed due to this manipulation. The colonies were collected after closing the lid at the midnight for individual marking and colony census. Adults emerging from the colonies were individually marked at 3-day intervals for behavioral observation, and every day for CHC analyses. There were few missing individuals that were foraging or off the nests because we marked every newly emerged individual at midnight. Behavioral observations in the field The behaviors of females (foraging, oviposition, dominance, and lateral vibration) in 12 queenright colonies were observed from late June to late July for 49 hours in 1993. Each of our observations was conducted for at least 55 minutes per day from 9 am to 3 pm by one person (KT) on sunny days. We counted the frequency of lateral vibration (wagging) separately from dominance acts, which are included in the category of dominance behavior (Gamboa and Dew 1981). Darting and biting opponents were classified as dominance behavior in this study. We counted occurrences of dominance behavior per individual because these behaviors are associated with reproduction in many paper wasps (e.g., West-Eberhard 1969; Jeanne 1972; De Souza and Prezoto 2012). We did not categorize or weight these dominance behaviors according to the level of escalation. We counted foraging trips that resulted in workers bringing water, honey, pulp, or flesh back to the nest. The data were analyzed using a log-linked Poisson general linear mixed model (GLMM), with the frequency of each variable of interest (lateral wagging, dominance act, foraging trip) and workers’ age (in days) included as an explanatory variable and the number of ovipositions as the response variable. Colonies and workers were treated as random effects, and observation time was treated as an offset variable. We used the lme4 package in R software (R Development Core Team) and selected the best models according to the Akaike information criterion (AIC). For GLMM analyses, the observation data were divided into four sessions with almost-identical day-period intervals according to colony development. The first session was before 7 July, the second was from 8 to 14 July, the third was from 15 to 21 July, and the fourth was from 22 July. The third and fourth sessions corresponded to the beginning of egg production by reproductive wasps of the next generation. A queen and her daughter workers were present in each colony in each session. Colony CHC analyses Each egg was collected using our handmade cell cups, which were previously inserted into empty cells to collect eggs without destruction or chemical contamination. The empty cells were produced by removal of the original eggs with fine forceps. The cell cup was made of filter paper, arranged using glue into the shape of a bucket without a bottom, by cutting off the tip of a triangular pyramid; using the inner wall of a 200-μL PCR tube, the filter paper was arranged in a pyramid, and the tip was cut off using scissors. The cups were immersed in anhydrous hexane (Sigma-Aldrich) for more than 24 hours. The cups were subsequently dried at room temperature and inserted into the empty cells. After oviposition on the cell cup by a wasp, the cup was gently withdrawn, and each egg was immersed in 50 μL of hexane for 24 hours at –20°C, after removing as much of the filter paper from around the egg as possible. The eggs were then removed from the hexane, and the remaining solution was kept at –20°C. Before hexane immersion, some of the collected eggs were gently placed within a controlled-temperature cabinet, maintained at 26°C ± 1°C with a 14L:10D photoperiod and 95% relative humidity, to determine the hatching rates. The differences in hatchability between the eggs produced by the queen and reproductive workers were analyzed using a log-linked binomial GLM. Colony was treated as a random effect. We collected and analyzed 220 eggs from 15 colonies from 2008 and 2010: we collected the eggs in both queenright and queenless conditions from eight of the colonies; we collected from five of the colonies under field conditions; and we collected eggs under laboratory conditions from ten of the colonies. At the end of our experiment (end of July), each adult was collected in an individual clean glass vial and stored at –80°C. CHCs were extracted by dripping on the adult body (abdomen) with 100 μL of hexane, and the resulting solution was stored at –20°C. The abdomens were dissected and the ovarian status was categorized into three groups: well-developed (more than two matured eggs), developed (one or two matured eggs), or undeveloped (zero matured eggs). The former two classes corresponded to reproductive workers, while the latter corresponded to non-reproductive workers. We collected eggs produced by both the queen and by reproductive workers, and under both queenright and queenless conditions. We collected body CHCs (n = 184) from the queen, reproductive workers, and non-reproductive workers from 15 queenright colonies. One-twelfth of the solution extracted from an egg, and 1/200 of that extracted from the body, were analyzed by gas chromatography-mass spectrometry (GC–MS) (HP6890; Hewlett-Packard) with an HP5973 mass spectrometer system (Hewlett-Packard) equipped with an HP-5MS column (0.25 mm ID × 30 m, 0.25 μm film thickness; Hewlett-Packard). The column temperature program for the former column was 150°C for 2 min, increasing by 7°C/min up to 325°C, followed by maintenance at this temperature for 6 min. The carrier gas was He. Identification of cuticular compounds was performed based on their mass spectra, as produced by electron impact ionization (70 eV). The solutions extracted from the eggs and abdomens were analyzed by a gas chromatography-flame ionization detector (GC–FID) (GC-17A ver. 3.0; Shimadzu) under the same conditions as described for GC-MS, and the temperature of the FID was 325°C. The area of the peak on each chromatograph was estimated using CLASS-GC10 software (Shimadzu). We calculated the proportional area of each peak to determine the total area of all peaks for each individual and egg, and analyzed CHCs only when the mean proportion exceeded 0.5%. We performed non-metric multidimensional scaling (NMDS) to visualize and analyze profiles of CHC differences between each egg and adult class. Permutational multivariate analysis of variance (PERMANOVA) was conducted to compare CHC profiles among classes using R software. We also analyzed CHC profiles using the random forest ensemble learning method, which applies decision trees to improve predictive performance (Breiman 2001), using R software. In these analyses, the relative importance of each CHC component among all CHCs was evaluated using the mean decrease in the Gini index. From these results, we extracted the five most important CHC components. Analysis of colonies under laboratory conditions Before the first worker emerged, 15 and 33 queenright nests were collected in the field around Gifu city in 2012 and 2013, respectively, and transported to the laboratory. The nests were attached to the wall of a cardboard box (30 × 30 × 60 cm). We cut lines 5 cm from the edge of each wall, except the wall to which the nest was attached (30 × 30 cm), and covered the remaining spaces with plastic film (Saran Wrap). The original foundress was released on the nest after weak immobilization on ice and fed with chopped mealworm (Tribolium castaneum) and silkworm (Bombyx mori) ad libitum. Tap water and honey were also supplied. The boxes were placed so that the top wall was 20 cm below a 20 W fluorescent light and maintained at 26°C ± 1°C with a 14L:10D photoperiod. Newly emerged adults discovered on daily inspection were collected, individually marked, and gently released back onto the nests. JH extraction and liquid chromatography–mass spectrometry (LC–MS) Each wasp in each class of workers, foundresses, and queens as described below was collected from the queenright colonies under room conditions and cooled on ice in 2013 and 2014. Hemolymph was subsequently collected by gently inserting a microcapillary tube between abdominal segments. After collection, the mouth of the remaining body was sealed with glue, centrifuged at 5,000 rpm for 5 min, and the remaining hemolymph was collected. The bodies were then dissected and the ovarian condition was checked. Only hemolymph that was clear yellow in color was used in further analysis. The amount of hemolymph collected from each wasp was measured, and JH was extracted from the samples following the methods of previous studies (Westerlund and Hoffman 2004; Cornette et al. 2008). Each hemolymph sample from 1 to 8 individuals was pooled prior to analysis. Totally, we used 172 individuals from 108 colonies for the extraction. Aliquots of 17 μL of hemolymph were homogenized in 400 mL methanol and allowed to stand at room temperature for 5 min. After centrifugation at 10,000 rpm for 1 min, the supernatant was collected and 30 ng fenoxycarb (Wako) was added as an internal standard. Then, 100 μL 2% NaCl solution and 300 μL hexane were added to the mixture, which was vortexed and allowed to stand at room temperature for 5 min. The mixture was centrifuged at 3,200 rpm for 5 min and the hexane phase was then transferred into a new glass vial. This process was repeated three times; finally, 900 μL hexane was obtained. The resulting mixture was stored at –80°C before vacuum drying in a CC-181 centrifugal concentrator (Tomy). Dried pellets were dissolved in 20 μL acetonitrile. LC–MS was performed using 20 μL each of concentrated sample, following the procedure described by Cornette et al. (2008). Aliquots of 5 μL were separated on a 150 × 2-mm2 inner diameter C18 reverse-phased column (YMC-Pack Pro C-18.5 μm; YMC Co., Ltd.) protected by a guard column (YMC-Pack Pro, sphere ODS; YMC Co., Ltd.) eluted with a gradient of water/methanol (80–100% methanol over 0–15 min, 100% methanol for 5 min) at a flow rate of 0.2 mL/min using an Agilent 1100 high-performance LC (HPLC) system with an autosampler (Agilent Technologies). Mass spectral analysis was performed by electrospray ionization in positive ion mode on a micro-high-resolution time of flight (TOF-HS) spectrometer (Bruker Daltonik) with the electrospray capillary set at 4.5 kV and under a drying temperature of 200°C. The nitrogen pressure of the nebulizer was 1.6 bar and the drying gas nitrogen flow rate was 9 L/min. Quantification of JH III and fenoxycarb was performed by monitoring the [M+H]+ and [M+Na] + ions. For each sample, a calibration curve for JH III (Sigma-Aldrich) was plotted using the same internal standard concentration as for fenoxycarb. The JH III titer from each sample was then calculated after analysis of the chromatogram data using QuantAnalysis software (Bruker Daltonik). Data are expressed as ng/wasp. JH titers were measured for the foundress, queen, and non-reproductive workers at 2 days of age, and again for non-reproductive and reproductive workers at 30 and 32 days of age, respectively. Topical application of JH and measurement of biogenic amine levels Two 0-day-old wasps were collected from each of 5 of 48 colonies in 2013 and 2014 under room conditions with forceps and immobilized on ice. One of the wasps was topically treated with 2 μL acetone as a control, and the other was treated with 2 μL JH III (Sigma-Aldrich) solution (50 μg/μL in acetone) using a micro-syringe. After the treatments, the wasps were gently released into their original colonies. The two wasps were collected again at 4- or 8-day, and immediately frozen with liquid nitrogen. The brains of the wasps were dissected out from the heads on a Peltier cooling unit under a dissecting microscope. The dissected brains were homogenized in a micro-glass homogenizer in 50 mL ice-cold 0.1 M perchloric acid containing 12.5 ng/mL 3,4-dihydroxybenzylamine (DHBA) as an internal standard. Each sample was then transferred into a 1.5-mL Eppendorf tube, and centrifuged at 15,000 rpm for 30 min at 0°C. The supernatants were transferred to micro-vials for analysis by HPLC-electrochemical detection (ECD). The HPLC system consisted of a solvent delivery pump (EP- 300; Eicom), a refrigerated automatic injector (231–401; Gilson), and a C18 reversed-phase column (UG 120; Shiseido) maintained at 35°C in a column oven. An electrochemical detector (ECD-300; Eicom) with a glassy carbon electrode (WE-GC; Eicom) was used. The detector potential was usually set at 0.87 V against an Ag/AgCl reference electrode. The detector cell was held at a constant temperature of 35°C by placing it in the column oven. Signals from the ECD were recorded and integrated using data analysis software (PowerChrom; ADInstruments). The mobile phase contained 0.18 M monochloroacetic acid and 40 mM 2Na-EDTA, which was adjusted to pH 3.6 with NaOH. Then, 1.62 mM of sodium-1-octanesulfonate was added into this solution as an ion-pair reagent and 7.4% CH3CN as an organic modifier. The mobile phase buffer was filtered through a 0.22-μm filter (Millipore) and degassed. The flow rate was kept constant at 0.7 mL/min. External standards were run before and after the sample runs. External standards [octopamine (OA), N-acetyldopamine (NADA), dopamine (DA), N-acetyltyramine (NATA), tyramine (TA), N-acetyl-5-hydroxytryptamine (NA5HT), tryptophan (TRP)], and serotonin [5-hydroxytryptamine (5HT)] were used for identification and quantification of biogenic amines. The peaks of each biogenic amine were identified by comparison of both the retention time and hydrodynamic voltammograms with those of the standards. Measurements based on the peak area of the chromatograms were obtained by calculating the ratio of the peak area of a substance to the peak area of the external standard. Amounts of biogenic amines were calculated in units of pmol/brain. We fitted GLMMs to clarify the effects of JH treatment and workers’ age (4 or 8 days old) on each monoamine. We presumed the objective variables, i.e., each monoamine, followed a normal (Gaussian) distribution with an identity link function. We set each colony and sample as the random intercept of all models. The analyses were performed using R software and the MCMCglmm library (ver. 2.24; Hadfield 2010), which is a package for fitting GLMMs using Markov Chain Monte Carlo (MCMC) techniques, and the estimate of each explanatory variable is given by its posterior distribution. We used 50,000 iterations after 5,000 burn-in iterations and set 2% as the thinning rate (1,000 samples were picked for the posterior distributions) to run the calculation. As MCMCglmm has no facility for setting multiple chains, we ran three MCMC calculations independently and the results were merged for calculation of R-hat. An R-hat value < 1.1 confirms the convergence of MCMC sampling for each explanatory variable (Gelman et al. 2004). We accepted an explanatory variable as being significant if the 95% confidence interval of its posterior distribution did not contain 0. RNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) The heads of fresh queens (n = 6), reproductive workers (n = 10), and non-reproductive workers (n = 10) taken from the colonies under room conditions in 2013 and 2014 were immersed in liquid nitrogen and stored at –80°C. Total RNA was extracted from the heads with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Briefly, each frozen head (kept at –80°C) of three adults’ castes was put into clean microtubes, and each head sample was roughly cut for filling with TRIzol reagent. Immediately, 300 μL of TRIzol reagent was added to each tube, and the samples were homogenized. The quality of total RNA was checked by spectrophotometry. For cDNA synthesis, 1 mg aliquots of total RNA were used in conjunction with the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). Each qRT-PCR mixture (12.5 μL) contained 0.5 μL of first-strand cDNA. Real-time detection and analysis of DNA were performed based on SYBR green dye chemistry using a SYBR Premix Ex Taq Perfect Real Time Kit (TaKaRa) and a Thermal Cycler Dice Real Time System (model TP700; TaKaRa). PCR was performed using primers for eight genes—insulin like-receptor 1 (ILR1), target of rapamycin (TOR), dopamine receptor D1 (DOP1), dopamine receptor D2 (DOP2), ultraspiracle (USP), vitellogenin (Vg), malvolio (malV), foraging (For)—designed based on the RNA-Seq data of Ferreira et al. (2013) or Toth et al. (2007). We selected these target genes because they are associated with social behavior (e.g., Ben-Shahar et al. 2003; Guidugli et al. 2005; Vergoz et al. 2007; Wheeler et al. 2014). The primer sequences are listed in Table S1. The mRNA values were normalized relative to ribosomal protein RPL37. The wasp bodies were dissected and for evaluation of ovarian development; here, the ovarian development index was calculated as the sum of the first- and second-ranking scores of maximum length × width among basal oocytes in the ovarioles in each pair of ovaries according to Sasaki et al. (2009). The index was used to distinguish non-reproductive workers (index <0.4) from reproductive workers (≥0.4) in our experiments on gene expression and JH. This index-based bifurcation was consistent with the ovarian status used in our behavioral observation, as workers with undeveloped eggs, those with developed eggs, and those with well-developed eggs were estimated to have average ovarian index values of 0.1, 0.6, and 1.7, respectively (data not shown).野外群落普查 于4月末至6月初首批职蜂羽化前，在野外采集附着于建筑物屋檐下的蜂后及蜂巢。每个蜂巢的柄用粘合剂（Konishi G17）垂直固定于带盖透明塑料盒（19×13×6 cm）的底部。对经个体标记的蜂后（所有群落均为单后建群巢穴）进行弱冰麻醉后，放归至原蜂巢。放归后，于午夜将塑料盒用金属丝固定于距原巢位点15 cm范围内。此次操作未观察到明显的蜂后丢失情况。次日午夜封闭盒盖后，收集该群落用于个体标记及群落普查。从群落中羽化的成虫每3天进行一次个体标记，用于行为观察；而用于表皮碳氢化合物（cuticular hydrocarbons, CHC）分析的个体则每日标记。由于我们于午夜对所有新羽化个体进行标记，因此极少出现因外出觅食或离巢而未被标记的个体丢失情况。 野外行为观察 1993年6月末至7月末，对12个有王群落中的雌性个体（觅食、产卵、等级支配及侧振动行为）进行了总计49小时的观察。每日的观察时长至少55分钟，由单人（KT）于晴天的9:00至15:00之间开展。我们将侧振动（摆尾）行为的发生频率与等级支配行为分开统计：后者属于支配行为范畴（Gamboa与Dew，1981）。本研究中，冲向并撕咬对手的行为被归类为等级支配行为。由于诸多马蜂物种的等级支配行为与繁殖相关，我们统计了每个个体的支配行为发生频次（West-Eberhard，1969；Jeanne，1972；De Souza与Prezoto，2012）。我们未根据行为升级程度对这些支配行为进行分类或赋权。我们统计了职蜂携带水、蜂蜜、木浆或肉类返回蜂巢的觅食行程次数。数据采用对数连接泊松广义线性混合模型（general linear mixed model, GLMM）进行分析：将各目标变量（侧摆尾行为、支配行为、觅食行程）的发生频次及职蜂日龄作为解释变量，产卵次数作为响应变量。将群落与职蜂作为随机效应，观察时长作为偏移变量。本研究使用R软件（R开发核心团队）中的lme4包，并根据赤池信息准则（Akaike information criterion, AIC）筛选最优模型。针对GLMM分析，我们根据群落发育进程将观察数据划分为四个间隔近乎一致的阶段：第一阶段为7月7日前，第二阶段为7月8日至14日，第三阶段为7月15日至21日，第四阶段为7月22日及以后。第三、第四阶段对应下一代繁殖型马蜂开始产卵的时期。每个阶段的每个群落中均存在蜂后及其雌性后代职蜂。 群落表皮碳氢化合物分析 采用自制巢杯采集蜂卵：提前将巢杯插入空巢房，以避免破坏巢房或造成化学污染。空巢房通过用细镊子移除原有的蜂卵获得。巢杯由滤纸制成：将滤纸按200 μL PCR管的内壁形状折叠为三角锥状，剪去尖端后用粘合剂固定为无底桶形。将巢杯置于无水己烷（Sigma-Aldrich）中浸泡24小时以上。随后将巢杯置于室温晾干，插入空巢房。当马蜂在巢杯上产卵后，轻轻取出巢杯，尽可能去除卵周围的滤纸后，将每个卵置于50 μL己烷中，于–20℃下浸泡24小时。随后将卵从己烷中取出，剩余的浸提液保存于–20℃。在浸入己烷前，将部分采集的蜂卵置于控温培养箱中（温度维持在26℃±1℃，光周期14L:10D，相对湿度95%）以测定孵化率。采用对数连接二项式广义线性模型（GLM）分析蜂后与繁殖型职蜂所产卵的孵化率差异，将群落作为随机效应。本研究于2008年和2010年从15个群落中采集并分析了220枚蜂卵：其中8个群落同时采集有王与无王条件下的卵；5个群落仅在野外条件下采集；10个群落仅在实验室条件下采集。实验结束时（7月末），将每只成虫收集至干净的单个玻璃小瓶中，保存于–80℃。通过向成虫（腹部）滴加100 μL己烷提取表皮碳氢化合物，所得浸提液保存于–20℃。解剖腹部并将卵巢状态分为三类：发育良好（成熟卵多于2枚）、发育中等（成熟卵1~2枚）及发育未成熟（无成熟卵）。前两类为繁殖型职蜂，最后一类为非繁殖型职蜂。我们采集了蜂后与繁殖型职蜂在有王及无王条件下所产的卵。从15个有王群落中采集蜂后、繁殖型职蜂与非繁殖型职蜂的表皮碳氢化合物样本（n=184）。取1/12的卵浸提液与1/200的成虫浸提液，通过气相色谱-质谱联用仪（GC-MS，HP6890；惠普公司）进行分析，该系统配备HP5973质谱检测器与HP-5MS色谱柱（内径0.25 mm×长30 m，膜厚0.25 μm；惠普公司）。色谱柱升温程序为：150℃保持2 min，以7℃/min的速率升至325℃，并维持该温度6 min。载气为氦气。表皮化合物的鉴定基于电子轰击电离（70 eV）所得的质谱图。卵与成虫腹部的浸提液同时采用气相色谱-火焰离子化检测器（GC-FID，GC-17A ver.3.0；岛津公司）分析，色谱条件与GC-MS一致，FID检测器温度为325℃。采用CLASS-GC10软件（岛津公司）计算每张色谱图的峰面积。计算每个峰的面积占比，以确定每个个体或卵的所有峰的总峰面积；仅当平均占比超过0.5%时，才对该表皮碳氢化合物组分进行分析。采用非度量多维标度（non-metric multidimensional scaling, NMDS）可视化并分析不同卵与成虫类群间的表皮碳氢化合物谱差异。采用R软件进行置换多元方差分析（permutational multivariate analysis of variance, PERMANOVA）以比较不同类群间的表皮碳氢化合物谱。同时采用随机森林集成学习方法（基于决策树以提升预测性能，Breiman，2001）通过R软件分析表皮碳氢化合物谱。此类分析中，通过基尼指数平均减少量评估每个表皮碳氢化合物组分在所有组分中的相对重要性。基于上述结果，筛选出5个最重要的表皮碳氢化合物组分。 实验室条件下的群落分析 首批职蜂羽化前，分别于2012年和2013年在岐阜市周边野外采集15个和33个有王蜂巢，并转运至实验室。将蜂巢附着于纸板箱（30×30×60 cm）的内壁。除蜂巢附着的30×30 cm内壁外，其余各壁均在距边缘5 cm处切割开口，并用保鲜膜（Saran Wrap）覆盖剩余空间。将创始蜂后经弱冰麻醉后放归蜂巢，并随意投喂切碎的黄粉虫（Tribolium castaneum）与家蚕（Bombyx mori）幼虫。同时提供自来水与蜂蜜。将纸板箱放置于顶部壁距20 W荧光灯20 cm处，维持环境温度26℃±1℃，光周期14L:10D。每日检查时发现的新羽化成虫将被收集、个体标记后轻轻放归蜂巢。 保幼激素（juvenile hormone, JH）提取与液相色谱-质谱联用（LC-MS） 2013年和2014年，从实验室条件下的有王群落中采集下述各类职蜂、创始蜂后及蜂后样本，置于冰上冷却。随后通过显微毛细管轻轻插入腹节间隙收集血淋巴。收集完成后，用粘合剂密封虫体断口，以5000 rpm离心5 min，收集剩余血淋巴。随后解剖虫体并检查卵巢状态。仅使用颜色清亮呈黄色的血淋巴进行后续分析。测定每只马蜂的血淋巴采集量，并按照已有研究方法（Westerlund与Hoffman，2004；Cornette等，2008）从样本中提取保幼激素。分析前将1~8只马蜂的血淋巴样本混合。本研究总计从108个群落中采集172只个体用于提取。将17 μL血淋巴等分试样在400 mL甲醇中匀浆，于室温静置5 min。以10000 rpm离心1 min后收集上清液，加入30 ng的苯氧威（Wako公司）作为内标。向混合物中加入100 μL 2%氯化钠溶液与300 μL己烷，涡旋混匀后于室温静置5 min。以3200 rpm离心5 min后，将己烷相转移至新的玻璃小瓶中。重复该步骤三次，最终收集到900 μL己烷提取物。将所得混合物保存于–80℃，随后使用CC-181离心浓缩仪（Tomy公司）进行真空干燥。将干燥后的沉淀物溶于20 μL乙腈。取20 μL浓缩后的样本按照Cornette等（2008）的方法进行LC-MS分析。取5 μL等分试样在带有保护柱（YMC-Pack Pro sphere ODS；YMC公司）的C18反相色谱柱（YMC-Pack Pro C18，5 μm；内径150×2 mm²；YMC公司）上分离，采用Agilent 1100高效液相色谱（HPLC）系统搭配自动进样器（Agilent科技公司），以0.2 mL/min的流速进行水/甲醇梯度洗脱（0~15 min内甲醇浓度从80%升至100%，100%甲醇维持5 min）。采用微高分辨飞行时间质谱仪（TOF-HS，Bruker Daltonik公司）以正离子电喷雾电离模式进行质谱分析：电喷雾毛细管电压设置为4.5 kV，干燥温度为200℃。雾化器氮气压力为1.6 bar，干燥气氮气流速为9 L/min。通过监测[M+H]+与[M+Na]+离子峰对JH III及苯氧威进行定量。每个样本均使用与苯氧威相同浓度的内标绘制JH III（Sigma-Aldrich）的标准曲线。采用QuantAnalysis软件（Bruker Daltonik公司）分析色谱数据后，计算每个样本中的JH III滴度。数据以ng/只马蜂为单位表示。分别测定2日龄创始蜂后、蜂后及非繁殖型职蜂的JH滴度，以及30日龄非繁殖型职蜂与32日龄繁殖型职蜂的JH滴度。 保幼激素局部施用与生物胺水平测定 2013年和2014年，从48个实验室群落中各选取5个群落，用镊子采集2只0日龄马蜂，置于冰上麻醉。其中一只马蜂以显微注射器局部施用2 μL丙酮作为对照，另一只施用2 μL JH III溶液（50 μg/μL，溶于丙酮，Sigma-Aldrich公司）。处理后将马蜂轻轻放归原群落。分别在4日龄或8日龄时再次收集这两只马蜂，立即用液氮冷冻。在体视显微镜下，于Peltier制冷台上从头部解剖出马蜂的大脑。将解剖出的大脑置于显微玻璃匀浆器中，用50 mL冰浴0.1 M高氯酸（含12.5 ng/mL 3,4-二羟基苄胺（DHBA）作为内标）进行匀浆。将每个样本转移至1.5 mL Eppendorf管中，于0℃以15000 rpm离心30 min。将上清液转移至微型样品瓶中，用于高效液相色谱-电化学检测（HPLC-ECD）分析。HPLC系统包括溶剂输送泵（EP-300；Eicom公司）、冷藏自动进样器（231-401；Gilson公司）及置于35℃柱温箱中的C18反相色谱柱（UG 120；Shiseido公司）。采用配备玻璃碳电极（WE-GC；Eicom公司）的电化学检测器（ECD-300；Eicom公司）。检测器电位通常设置为相对于Ag/AgCl参比电极的0.87 V。将检测器池置于柱温箱中，维持恒定温度35℃。采用数据分析软件（PowerChrom；ADInstruments公司）记录并积分ECD信号。流动相包含0.18 M一氯乙酸与40 mM 2Na-EDTA，用NaOH调节至pH 3.6。向该溶液中加入1.62 mM 1-辛烷磺酸钠作为离子对试剂，以及7.4%的乙腈作为有机改性剂。将流动相缓冲液通过0.22 μm滤膜（Millipore公司）过滤并脱气。流速维持在0.7 mL/min。在样本分析前后均运行外标样本。采用外标物[章鱼胺（OA）、N-乙酰多巴胺（NADA）、多巴胺（DA）、N-乙酰酪胺（NATA）、酪胺（TA）、N-乙酰-5-羟色胺（NA5HT）、色氨酸（TRP）]与5-羟色胺（5HT）对生物胺进行定性与定量分析。通过将各生物胺的峰保留时间与伏安图与标准品对比进行鉴定。通过计算目标物质峰面积与外标峰面积的比值，得到基于色谱峰面积的定量结果。生物胺含量以pmol/大脑为单位计算。采用广义线性混合模型（GLMM）分析JH处理与职蜂日龄（4或8日龄）对各单胺类物质的影响。假设目标变量（各单胺类物质）服从正态（高斯）分布，采用恒等连接函数。将每个群落与样本设置为所有模型的随机截距项。本分析采用R软件与MCMCglmm包（版本2.24；Hadfield，2010）进行：该包通过马尔可夫链蒙特卡洛（MCMC）技术拟合GLMM，各解释变量的估计值由其后验分布给出。计算时采用5000次预迭代后运行50000次迭代，并设置2%的抽稀率（从后验分布中抽取1000个样本）。由于MCMCglmm无法设置多条马尔可夫链，我们独立运行三次MCMC计算并合并结果以计算R-hat值。当R-hat值<1.1时，可确认各解释变量的MCMC采样收敛（Gelman等，2004）。若某解释变量的后验分布95%置信区间不包含0，则认定其具有统计学显著性。 RNA提取与定量反转录聚合酶链式反应（qRT-PCR） 2013年和2014年，从实验室群落中采集新鲜的蜂后（n=6）、繁殖型职蜂（n=10）与非繁殖型职蜂（n=10）的头部，浸入液氮后保存于–80℃。按照试剂盒说明书，使用TRIzol试剂（Life Technologies公司）从头部组织中提取总RNA。简要步骤如下：将保存于–80℃的三种成虫类群的冷冻头部置于干净的微量离心管中，粗略切碎后加入TRIzol试剂。每管立即加入300 μL TRIzol试剂，对样本进行匀浆。通过分光光度法检测总RNA的质量。采用PrimeScript RT试剂盒（带有gDNA清除剂，TaKaRa公司），以1 mg总RNA为模板进行cDNA合成。每个qRT-PCR反应体系（12.5 μL）包含0.5 μL第一链cDNA。采用SYBR Premix Ex Taq Perfect Real Time试剂盒（TaKaRa公司）与Thermal Cycler Dice实时荧光定量系统（TP700型；TaKaRa公司），基于SYBR Green染料化学法进行实时DNA检测与分析。基于Ferreira等（2013）与Toth等（2007）的RNA测序数据设计8个基因的引物：胰岛素样受体1（ILR1）、雷帕霉素靶蛋白（TOR）、多巴胺受体D1（DOP1）、多巴胺受体D2（DOP2）、超螺旋蛋白（USP）、卵黄原蛋白（Vg）、malvolio基因（malV）与觅食基因（For）。选择这些靶基因是因为它们与社会行为相关（Ben-Shahar等，2003；Guidugli等，2005；Vergoz等，2007；Wheeler等，2014）。引物序列详见附表S1。以核糖体蛋白RPL37作为内参基因对mRNA表达量进行标准化。解剖马蜂虫体以评估卵巢发育情况：按照Sasaki等（2009）的方法，卵巢发育指数计算为每对卵巢的小管中基部卵母细胞前两名最大长度×宽度的总和。在本研究的基因表达与JH实验中，通过该指数区分非繁殖型职蜂（指数<0.4）与繁殖型职蜂（指数≥0.4）。该指数划分结果与本研究行为观察中所用的卵巢状态一致：卵发育未成熟、发育中等及发育良好的职蜂的平均卵巢指数分别为0.1、0.6和1.7（未展示数据）。