Apidologie (2019) 50:793–803 Original article * INRA, DIB and Springer-Verlag France SAS, part of Springer Nature, 2019 DOI: 10.1007/s13592-019-00686-9 The capping pheromones and putative biosynthetic pathways in worker and drone larvae of honey bees Apis mellifera 1,2 1 3 4 1 Qiu-Hong QIN , Xu-Jiang HE , Andrew B. BARRON , Lei GUO , Wu-Jun JIANG , 1 Zhi-Jiang ZENG 1Honeybee Research Institute, Jiangxi Agricultural University, Nanchang 330045 Jiangxi, People’s Republic of China 2Guangxi Liuzhou Animal Husbandry and Veterinary School, Liuzhou 545003 Guangxi, People’s Republic of China 3Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia 4Research and Development Centre, China Tobacco Jiangxi Industrial Co., LTD., Nanchang 330096 Jiangxi, People’s Republic of China Received 24 January 2019 – Revised 28 June 2019 – Accepted 1 September 2019 Abstract – In honey bees (Apis mellifera ), methyl palmitate (MP), methyl oleate (MO), methyl linoleate (ML), and methyl linolenate (MLN) are important pheromone components of the capping pheromones triggering the capping behavior of worker bees. In this study, we compared the amounts of these four pheromone components in the larvae of workers and drones, prior to be capped, in the process of being capped and had been capped. The amounts of MP, MO, and MLN peaked at the capping larval stage, and ML was highest at capped larvae in worker larvae, whereas in drone larvae, the amounts of the four pheromone components were higher overall and increased with aging. Furthermore, we proposed de novo biosynthetic pathways for MP, MO, ML, and ML, from acetyl-CoA. Besides, stable isotope tracer 13C and deuterium were used to confirm that these capping pheromone components were de novo synthesized by larvae themselves rather than from their diets. honey bee / larvae / capping / pheromones / pathways 1. INTRODUCTION mones help to coordinate a colony’s collective behaviour, such as foraging, defensive behaviour, Honey bees have been instrumental in reveal- and brood-rearing activity (Free and Winder ing both the significance and the complexity of 1983; Free 1987; Vallet et al. 1991; Breed et al. pheromonal communication systems. Phero- 2004; Hunt 2007; Stout and Goulson 2001; Slessor et al. 2005; Maisonnasse et al. 2010). Electronic supplementary material The online version of The subtleties and complexities of bee pheromon- this article (https://doi.org/10.1007/s13592-019-00686-9) al communication are perhaps best illustrated by contains supplementary material, which is available to the pheromonal communication between workers authorized users. and developing larvae. Corresponding author: Z. Zeng, [email protected] Honey bee larvae are entirely dependent on Qiu-Hong Qin and Xu-Jiang He contributed equally to their attendant adult workers. Larvae release cap- this work. ping pheromone components, form their salivary All sequencing data has been submitted to GenBank under accession numbers SRR4051855, SRR4051820, glands containing ten methyl or ethyl fatty acid bioproject PRJNA339509 esters (Le Conte et al. 1989, 1990, 2006;Trouiller Handling editor: Klaus Hartfelder et al. 1991). The composition of this pheromone 794 Q.-H. Qin et al. varies with larval age and sex (worker or drone), under the wax cover, which were approximately and workers adjust their behavioral responses to 8-day-old instar for worker and 9-day-old instar larvae accordingly (Free and Winder 1983;Le for drones. There were four biological replicates Conte 1994; Slessor et al. 2005). When larvae of each group, with each replicate sourced from a reach the stage of pupation, their cells are closed different colony. Each sample contained groups of off by workers constructing a thin wax cap over larvae to provide enough biological material for the cell so that the larvae pupate in a clean and gas chromatograph-mass spectroscopy (GC/MS) stable environment. Four of the components of analysis. For each sample, we collected 40 4th BEP are particularly important in triggering the instar worker and drone larvae, 20 capping-stage capping behaviour of workers. These are methyl and capped worker larvae, and 5 capping-stage palmitate (MP), methyl oleate (MO), methyl lino- and capped drone larvae from brood frames. leate (ML), and methyl linolenate (MLN) (Le The whole larvae of each sample group freshly Conte et al. 1990). Production of these compo- collected from their wax cells were placed in nents by larvae increases quite dramatically when (without body washes) glass vials with 3 ml di- the larva reaches the developmental stage prior to chloromethane, respectively. Twenty microliters pupation (Trouiller et al. 1991). of methyl nonadecanoate (≥ 98%, AccuStandard, Currently, our knowledge of how pheromones 10 μg/ml) was added to each vial as the internal are synthesized and released by worker and drone standard. Afterwards, the glass vials with larvae larvae at different developmental stages are limit- were shaken lightly for 30 min using an oscillator ed. To fulfill the gaps, here we examined changes (120 r/min), and then, the solution was transferred in the BEP components related to cell capping in into a clean vial and concentrated under nitrogen both worker and drone larvae pre, during and flow to about 20 μl. A 1-μl sample was injected post-cell capping. In parallel, we analyzed gene into the GC/MS. expression changes in larvae across the cell cap- A GC/MS system (7890A/5975C, Agilent ping stage as a first step to the identification of Technologies Inc., Santa Clara, CA, USA) with biosynthetic pathways in bees for pheromone an HP-Innowax chromatographic column (60-m, components. 0.25-mm, 0.25-μm film thickness, 19091N-136, Agilent Technologies) was used. Helium was used 2. MATERIALS AND METHODS as carrier gas at a constant flow rate of 1.0 ml/min. Samples were analyzed with time split injection 2.1. Experimental honey bee colonies mode and split ratio of 2:1. The column tempera- ture was programmed as follows: 120 °C for Honey bee colonies (Apis mellifera )used 1 min, then rising to 180 °C at a rate of throughout this study were maintained at the Hon- 5 °C/min, and then rising to 230 °C at a rate of eybee Research Institute, Jiangxi Agricultural 2 °C/min, finally cooling to 250 °C at a rate of University, Nanchang, China (28.46° N, 115.49° 5 °C/min and held constant for 10 min. The tem- E), according to standard beekeeping techniques. perature of the sample injection port was 250 °C, and the pressure of helium was 13.5552 psi. Sam- 2.2. Gas chromatograph-mass spectroscopy ple ionization was performed in electron impact analysis ionization mode at 70 eV. The temperature of ion source, electrode stem, and transmission line were Amounts of capping pheromone components 250 °C, 150 °C, and 250 °C, respectively. Single- were compared in the following sample groups: ion monitoring chromatograms were reconstruct- (i) non-capped larvae of 4-day-old (4th) instar of ed at the base peak for MP, MO, ML, and MLN workers and drones; (ii) capping-stage worker and and the internal standard: mass-to-charge ratio drone larvae sampled as wax caps were being (m/z) 270, 264, 294, 292, and 312. constructed over their cells; (iii) capped worker For each compound of interest, peak areas were and drone larvae sampled from cells that had been converted to amounts by reference to calibration capped completely and had a thin silk cocoon curves for each compound. MP, MO, ML, and The capping pheromones and putative biosynthetic pathways in worker 795 MLN (≥ 99.5%, Sigma) were used as external KEGG Orthology Based Annotation System standards. Seven concentrations of each external 2.0 (KOBAS 2.0). The KOBAS 2.0 (Xie standard (0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, et al. 2011) references the provided gene 2 μg/ml, 5 μg/ml, 10 μg/ml, and 25 μg/ml) were database against multiple existing databases used to construct calibration curves. All of the of metabolic and signaling pathways to iden- correlation coefficients of the four calibration tify pathways that are enriched in the RNA- curves were > 99.96% (Figure S1), which sug- seq samples. gested that the GC/MS system was stable enough for the later quantitative analysis of MP, MO, ML, 2.5. Verification of DEGs with qRT-PCR and MLN from honey bee larvae. analysis 2.3. RNA-Seq data analysis Six genes, which participate in the inferred biosynthetic pathways of MP, MO, ML, and Samples from the groups described above were MLN (Table SI)anda“housekeeping gene” also used for RNA-sequence analysis to analyze (β-actin), were selected for qPCR analysis. differences in gene expression between groups. For each group, 3 larvae from each of three For each sample, we collected six workers or colonies were sampled, yielding three biolog- drone larvae and created three biological repli- ical replicates per group. Samples were flash cates with each replicate sourced from a different frozeninliquidnitrogenandstoredat− colony. The RNA was extracted with Trizol. The 80 °C until used. Detailed methods for qRT- samples were flash frozen in liquid nitrogen and PCR are described in our previous study stored at − 80 °C until use. (Qin et al. 2014). Primers for these genes RNA extraction, RNA sequencing, and data were designed by Primer 5.0 (Table SI). For analysis were performed according to the methods each gene, three technical replicates were described in He et al. (2016). Briefly, the raw performed for each sample. The Ct value counts for each gene were normalized with Frag- for each sample was obtained by calculating ments Per Kilobase of transcript per Million the mean of three technical replicates. Rela- (FPKM) to calculate the P value for significantly tive expression levels between samples were regulated genes with DESeq packages.