Feeding Asian honeybee queens with European honeybee royal jelly alters body color and expression of related coding and non-coding RNAs

Abstract

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.

Roles of DNA Methylation in Color Alternation of Eastern Honey Bees (Apis cerana) Induced by the Royal Jelly of Western Honey Bees (Apis mellifera)

“…Of the 1020 DMGs, 20 DMGs were enriched into four key pathways (tryptophan, tyrosine, dopamine and phenylalanine; see Figure 3 B and Table S7 ), which are key pathways for honey bee body color formation. We compared the DMGs with DEGs from our previous study [ 12 ]. Interestingly, three DMGs were the same as the DEGs ( ALDH7A1 , ALDH2 , OGDH ) that are enriched in the tryptophan pathway.…”

“…Interestingly, three DMGs were the same as the DEGs ( ALDH7A1 , ALDH2 , OGDH ) that are enriched in the tryptophan pathway. The tryptophan pathway is vital for the formation of honey bee body color, resulting in an alternation from black in CQ control queen to yellow in NQ queens [ 12 ]. Our data clearly show that the various methylation sites of these three key DMGs [ OGDH , ALDH(NAD+) and ALDH7 ] differed between NQs and CQs, specifically those marked in red sites in ( Figure 4 A–C), reflecting the notable changes in DNA methylation of those key genes induced by A. cerana–A.…”

“…mellifera nutritional crossbreed could alter the genome-wide alternative splicing of A. cerana [ 11 ]. Recently, our previous findings revealed that LncRNAs and miRNAs play a key role in the full nutritional crossbreed of A. cerana and A. mellifera by regulating the expression of key genes of melanin biosynthesis pathways resulting in an alternation in the body color of A. cerana queens [ 12 ]. These findings emphasize that body color alternation is one of the most noticeable occurrences resulting from A. cerana–A.…”

“…Here, we hypothesized that the honey bee’s nutritional crossbreed induced by different larval diets from distinct honey bee species would be possibly controlled by epigenetic factors. Consequently, DNA methylation, as non-coding RNAs [ 12 ], would also contribute to the regulations of phenotypic alternation in A. cerana–A. mellifera nutritional crossbreed.…”

“…Previous evidence has shown that exchanging the larval diets between A. cerana and A. mellifera results in a clear body color alternation [ 7 , 8 , 12 , 26 ], and some non-coding RNAs are involved in this color alternation via regulating the expression of key genes which are related to the melanin synthesis [ 12 ]. However, it is still unclear whether other epigenetic modifications such as DNA methylation are involved in the regulation of those key genes.…”