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.

Keywords: body color alteration; gene expression; honeybees; non-coding RNA expression; nutritional crossbreed.

FIGURE 1

(A)The body color alternation in Apis cerana-Apis mellifera nutritional crossbreed. The 1-day worker larvae from their A. cerana mother queen were fed with A. mellifera royal jelly (AmRJ) based diet or A. cerana royal jelly (AcRJ), resulted in yellow color nutritional crossbreed queens (NQ) or black color control queens (CQ) respectively. (B) The body color quantification had significant differences of (NQs, n = 4, Scutum’s M = 2.5, SD = 1.29, t = 0.85, Scutellum’s M = 8.9, SD = 0.81, t = 5.84, Tergum’s M = 8.5, SD = 1.25, t = 63.81, and Sternum’s M = 9.5, SD = 0.95, t = 15.4) compared to (CQs, n = 4, Scutum’s M = 1.83, SD = 0.62 t = 5.11, Scutellum’s M = 0.81, SD = 0.62, t = −16.12, Tergum’s M = 2.8, SD = 0.62 t = 63.81, and Sternum’s M = 5.8, SD = 0.92, t = 15.14) all P were < 0.05) based on Ruttner’s color scales. Bars present as values of Mean ± SD, the black dots on the top of each bar represent replicates. Different letters on the top of bars indicate significant difference (p < 0.05, Independent-sample t-test).

FIGURE 2

(A) The volcano diagram of lncRNAs between NC and NQ. The red spots represent upregulated DElncRNAs in NQ compared to CQ, whereas the green spots represent downregulated DElncRNAs; The black spots are non-different lncRNAs. (B) The volcano diagram of DEGs between NC and NQ. The red spots represent upregulated DEGs in NQ compared to CQ, whereas the green spots represent downregulated DEGs. lncRNAs with FDR < 0.05, |log2 (Fold change) |≧1 were identified as DElncRNAs. Same to DEGs, DEmiRNAs and DEcircRNAs. (C) The volcano diagram of miRNAs between NC and NQ. The red spots represent upregulated DEmiRNAs in NQ, whereas green spots represent downregulated DEmiRNAs. (D) The volcano diagram of circRNAs between NC and NQ. The red spots represent upregulated DEcircRNAs in NQ, whereas green spots represent downregulated DEcircRNAs. (E–H) are the top pathways of KEGG enrichment of DEGs, DElncRNAs, DEmiRNAs and DEcircRNAs respectively. The sizes of circles represent the number of DEGs, DElncRNAs, DEmiRNAs, and DEcircRNAs, and the colors of circles represent the p-values of enrichment.

FIGURE 3

(A) The predicted KEGG network of honeybee body color alternation in nutritional crossbreed. This network is based on four key KEGG pathways including tyrosine, tryptophan, dopamine, and phenylalanine pathways. The key DEGs, DElncRNAs and DEmiRNAs involved in this network are also presented. Yellow bars represent DElncRNAs, sky blue bars represent DEmiRNAs, and purple bars represent DEGs and green bars represent key genes but not DEGs. Blue arrows mean DElncRNAs or DEmiRNAs involved into the regulation of related genes. (B) The heatmap of key DEGs, DElncRNAs and DEmiRNAs are involved into three key KEGG pathways. Different colors represent significantly differential expression of key DEGs, DElncRNAs and DEmiRNAs using their log2 (Fold change) values. Olive green arrows represent the regulatory relationship between key DEGs and non-coding RNAs (DElncRNAs and DEmiRNAs).

FIGURE 4

(A) The expression of TPH1 and KMO genes in 4-day A. cerana queen larvae by RNAi. Bars represent mean ± SD values of relative gene expression. Different letters on the top of each bar represent the significant difference (p < 0.05, independent-sample t-test). (B) The body color alternation of A. cerana queens in RNAi experiment. The upper queens with a yellow body color are TPH1-RNAi group, the middle ones are KMO RNAi group with slight body color changes compared to control bees. The lower ones are control queens fed with negative siRNA. (C)The body color quantification of KMO-iRNA (n = 4), TPH1-iRNA (n = 4), and control queens (n = 4) based on Ruttner’s color scales. Bars are present as values of Mean ± SD. Different letters on the top of the bars indicate significant differences (p < 0.0001 F = 25.41 F critical = 4.25, One-Way ANOVA), the same letter indicates no significant difference, and the black dots on the top of each bar represent replicates.