Molecular Ecology (2017) 26, 1598–1607 doi: 10.1111/mec.13990 Making a queen: an epigenetic analysis of the robustness of the honeybee (Apis mellifera) queen developmental pathway XU JIANG HE,* LIN BIN ZHOU,* QI ZHONG PAN,* ANDREW B. BARRON,† WEI YU YAN* and ZHI JIANG ZENG* *Honeybee Research Institute, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China, †Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia Abstract Specialized castes are considered a key reason for the evolutionary and ecological suc- cess of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honeybee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epige- netically regulated divergent developmental pathways. This is an important phe- nomenon in understanding the evolution of social insect societies. Here, we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3 days age, but queens reared from older worker larvae had decreased queen body size and weight compared with queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3-day-old queen larvae raised from worker larvae compared with that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen–worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates. Keywords: DNA methylation, epigenetic analysis, gene expression, honeybee, immunity, queen Received 7 March 2016; revision received 12 December 2016; accepted 19 December 2016 (Queller & Strassmann 1998; Linksvayer & Wade 2005; Introduction Foster et al. 2006). Oster and Wilson have particularly The evolution of cooperation, cooperative living and emphasized the importance of caste in the evolution of animal societies has been an enduring subject of fasci- social insect societies (Oster & Wilson 1978). Different nation for evolutionary biologists (Wilson 1975). Key castes within the society specialize on different func- insights into the processes of social evolution have tions. This specialization promotes efficiencies, which come from studies of the advanced social insects (Eilson provides a key selective advantage to social living. 1971; Andersson 1984; Robinson 1999). These have Oster & Wilson (1978) argue castes are one key reason shaped our understanding of the genetic and ecological for the ecological success of the social insect lifestyle. factors that can promote the evolution of sociality Queens and workers are the defining caste distinction for the social insects. Queens have multiple morpholog- ical and behavioural specializations for extreme fecun- Correspondence: Zhi Jiang Zeng, Fax: +86 791 83828176; dity, whereas workers show a similar degree of E-mail: [email protected] © 2016 John Wiley & Sons Ltd EPIGENETIC CHANGES IN HONEYBEE QUEEN REARING 1599 specialization for social roles supporting the queens’ and space) changes induced by the commercial rearing reproduction, and in many social insects workers are practice may potentially affect queen development via sterile. This is the case for honeybees (Apis mellifera). A epigenetic processes. Here, we explored the conse- typical colony contains a single reproductive queen sup- quence of age of transplant from worker cells to queen ported by up to 50 000 sterile workers (Winston 1991). cells on DNA methylation, gene expression and queen Studies of the bee have shown how the distinction morphology. We found that the domestic rearing prac- between queens and workers is not genetic: rather these tice altered queen morphology and induced epigenetic two phenotypes are the outcome of different develop- changes in developing queens, which supports our mental pathways (Nijhout 2003; Linksvayer et al. 2011). hypothesis. Both queens and workers develop from fertilized eggs, but differences in nutrition and the amount of Materials and methods food given to young larvae trigger different epigeneti- cally regulated developmental pathways (Kucharski Three European honeybee colonies (Apis mellifera) each et al. 2008; Maleszka 2014; Maleszka et al. 2014). with a single drone inseminated queen (SDI) were used Kucharski et al. (2008) reported that nutritional differ- throughout this study. These colonies were maintained ences between queen and worker at their larval stage at the Honeybee Research Institute, Jiangxi Agricultural control their development via DNA methylation. Shi University, Nanchang, China (28.46 uN, 115.49 uE), et al. (2011) showed that the amount of space in which according to the standard beekeeping techniques. a larva can develop alters the DNA methylation level of the larval genome and contributes to the process of Queen-rearing methods caste differentiation. Changes in gene regulation caused by these epigenetic mechanisms then establish diver- Queens were restricted for 6 h to a plastic honeybee gent developmental paths (Simola et al. 2013), involving frame developed by Pan et al. (2013) for laying. The particularly genes involved in signal transduction, frame is designed such that the plastic base of this gland development and carbohydrate metabolism frame with eggs or larvae can be transferred to plastic (Woodard et al. 2011). queen cells directly (Pan et al. 2013). Eggs that queen Since the 19th century in commercial beekeeping, it laid in worker cells were transplanted into queen cells has been a standard practice to raise queens by trans- for rearing new queens when eggs were less than 6 h planting eggs or young larvae into artificial queen cells, old (QWE). For the other experimental groups, day 1, which triggers workers to raise a queen (Doolittle 1888; day 2 and day 3 worker larvae were transplanted into Buchler€ et al. 2013). Within the commercial queen-rear- queen cells for rearing QWL1, QWL2 and QWL3, ing practice, there is variation in the age at which eggs respectively. Queen cells with worker eggs or larvae or worker larvae are transplanted to queen cells to be were returned into their natal colonies (the SDI colo- raised as queens. It is not clear how well the honeybees’ nies) for queen rearing. developmental processes are able to tolerate this kind For the morphological measurements, new emerging of intervention. Woyke (1971) reported that rearing queens were collected and their weight measured using queens from young worker larvae resulted in decreased an analytical balance (FA3204B; Shanghai Precision Sci- body size, a smaller spermatheca and fewer ovarioles. entific Instrument Co., Ltd.). Their thorax width and Rangel et al. (2012) reported that colonies from queens length were measured with a zoom stereo microscope reared from older worker larvae had significantly lower system (Panasonic Co., Ltd.) according to the manufac- production of worker comb, drone comb and stored turer’s instructions. food compared with colonies from queens reared from For epigenetic analysis, we sampled 3-day-old larvae young worker larvae. In fact, concern over the long- from QWE, QWL1 and QWL2, respectively, from their term consequences of commercial queen rearing for bee queen cell. The fourth group QWL3 sampled 3-day-old stocks is not new. In 1923, Rudolf Steiner predicted that worker larvae directly from worker cells. Each sample honeybees would become extinct within 100 years as a group collected three larvae and there were three bio- consequence of commercial queen rearing progressively logical replicates, each from different colonies, for each weakening bee stocks (Thomas 1998). In the current group. We weighed each larva from these four treat- environment of increased honeybee colony failure rates, ment groups with an analytical balance. All samples mass deaths of colonies and declining honeybee stocks, were immediately flash-frozen in liquid nitrogen. The there is a great deal of concern as to whether a decline DNA and RNA from each sample were both extracted in queen bee quality might be a factor in these prob- for further DNA methylation and RNA sequencing lems (van Engelsdorp et al. 2010; Delaney et al. 2011). analysis. DNA and RNA were extracted from the same Therefore, we hypothesize that environmental (nutrition samples. © 2016 John Wiley & Sons Ltd 1600 X. J. HE ET AL. < RNA-Seq analysis KEGG protein database by BLAST (E-value 1e-5) and used KOBAS 2.0 software to test the statistical enrichment Total RNA was extracted from larvae according to the of differential expression genes in KEGG pathways (Xie standard protocol for the TRIzol reagent (Life technolo- et al. 2011). gies, California, USA). RNA integrity and concentration were checked using an Agilent 2100 Bioanalyzer (Agi- DNA Methylation analysis by bisuphite sequencing lent Technologies, Inc., Santa Clara, CA, USA). mRNA was isolated from total RNA using a NEB- The DNA of each larval sample was