The queen and worker castes of the honey bee are very distinct phenotypes that result from different epigenomically regulated developmental programs. In commercial queen rearing, it is common to produce queens by transplanting worker larvae to queen cells to be raised as queens. Here, we examined the consequences of this practice for queen ovary development and genome-wide methylation. Queens reared from transplanted older worker larvae weighed less and had fewer ovarioles than queens reared from transplanted eggs. Methylome analyses revealed a large number of genomic regions in comparisons of egg reared and larvae reared queens. The methylation differences became more pronounced as the age of the transplanted larva increased. Differentially methylated genes had functions in reproduction, longevity, immunity, and metabolic functions suggesting that the methylome of larval reared queens was compromised and more worker-like than the methylome of queens reared from eggs. These findings caution that queens reared from worker larvae are likely less fecund and less healthy than queens reared from transplanted eggs.

Queen-worker caste dimorphism is a typical trait for honeybees (Apis mellifera). We previously showed a maternal effect on caste differentiation and queen development, where queens emerged from queen-cell eggs (QE) had higher quality than queens developed from worker cell eggs (WE). In this study, newly-emerged queens were reared from QE, WE, and 2-day worker larvae (2L). The thorax size and DNA methylation levels of queens were measured. We found that queens emerging from QE had significantly larger thorax length and width than WE and 2L. Epigenetic analysis showed that QE/2L comparison had the most different methylated genes (DMGs, 612) followed by WE/2L (473), and QE/WE (371). Interestingly, a great number of DMGs (42) were in genes belonging to mTOR, MAPK, Wnt, Notch, Hedgehog, FoxO, and Hippo signaling pathways that are involved in regulating caste differentiation, reproduction and longevity. This study proved that honeybee maternal effect causes epigenetic alteration regulating caste differentiation and queen development.

Background and aims: The Asian honeybee (Apis cerana) and the European honeybee (Apis mellifera) are reproductively isolated. Previous studies reported that exchanging the larval food between the two species, known as nutritional crossbreeding, resulted in obvious changes in morphology, physiology and behavior. This study explored the molecular mechanisms underlying the honeybee nutritional crossbreeding.Methods: This study used full nutritional crossbreeding technology to rear A. cerana queens by feeding them with an A. mellifera royal jelly-based diet in an incubator. The body color and the expression of certain genes, microRNA, lncRNA, and circRNA among nutritional crossbred A. cerana queens (NQ), and control A. cerana queens (CQ) were compared. The biological functions of two target genes, TPH1 and KMO, were verified using RNA interference.Results: Our results showed that the NQ’s body color turned yellow compared to the black control queens. Whole transcriptome sequencing results showed that a total of 1484, 311, 92, and 169 DEGs, DElncRNAs, DEmiRNAs, and DEcircRNAs, respectively, were identified in NQ and CQ, in which seven DEGs were enriched for three key pathways (tryptophan, tyrosine, and dopamine) involved in melanin synthesis. Interestingly, eight DElncRNAs and three DEmiRNAs were enriched into the key pathways regulating the above key DEGs. No circRNAs were enriched into these key pathways. Knocking down two key genes (KMO and TPH1) resulted in altered body color, suggesting that feeding NQ’s an RNAi-based diet significantly downregulated the expression of TPH1 and KMO in 4-day-old larvae, which confirmed the function of key DEGs in the regulation of honeybee body color.Conclusion: These findings reveal that the larval diets from A. mellifera could change the body color of A. cerana, perhaps by altering the expression of non-coding RNAs and related key genes. This study serves as a model of epigenetic regulation in insect body color induced by environmental factors.

Queens and workers are very distinct phenotypes that develop from the same genome. Larvae from worker cells up to 3.5 d old can be transferred to larger queen cells and will subsequently be reared as queens and develop into functional queens. This has become a very popular queen rearing practice in contemporary apiculture. Here we used RNA-Seq to study the consequences of rearing queens from transplanted worker larvae on the transcriptome of the adult queens. We found that queens reared from transferred older larvae developed slower, weighted less, and had fewer ovarioles than queens reared from transferred eggs, indicating queens were cryptically intercaste. RNA-Seq analysis revealed differentially expressed genes between queens reared from transferred larvae compared with queens reared from transferred eggs: the older the larvae transferred, the greater the number of differentially expressed genes. Many of the differentially expressed genes had functions related to reproduction, longevity, immunity, or metabolism, suggesting that the health and long-term viability of queens was compromised. Our finds verify the previous studies that adult queens reared from older transferred larvae were of lower quality than queens reared from transferred eggs or younger larvae.

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