*3.3. Differences in Royal Jelly Fatty Acid Composition*

In the present study, a relative quantitative analysis of SFAs, MUFAs, and PUFAs was carried out in royal jelly. Trends in levels of SFAs, MUFAs, and PUFAs were similar to those of SMs, Cers, PCs, and PEs (Figure 2). Specifically, SFA, MUFA, and PUFA contents were significantly decreased in royal jelly after initial feeding with single bee pollen. However, as honeybees adapted to the single-pollen diet, levels of SFAs, MUFAs, and PUFAs gradually increased. The highest levels were on day 18 or 24, but levels decreased on day 30.

In addition, trends in 10-hydroxydec-2-enoic acid (10-HDA) levels were consistent with those of other lipids in royal jelly (Figure 2): 10-HDA content was significantly decreased after initial feeding with single bee pollen; levels gradually increased as honeybees adapted to the single-pollen diet; the highest level of 10-HDA occurred on day 18 or 24; and levels were decreased on day 30. The average 10-HDA content was significantly higher in RJ-Am than in RJ-Pa. These results indicated that continuous feeding of different types of single bee pollen had significant effects on 10-HDA content in royal jelly.

Furthermore, we compared the relative abundance of 18-carbon fatty acids in bee pollen and found that the content of FA(18:0) and FA(18:1) in BP-Am was significantly higher than that in BP-Pa (Table 1). However, there's no significant difference in the relative abundance of 18-carbon fatty acids between RJ-Am and RJ-Pa (Figure 3), which might be attributed to the participation of 18-carbon fatty acids in lipid metabolism and the synthesis of 10-HDA. We also analyzed the ratio of free and bound 18-carbon saturated and unsaturated FAs in RJ-Am and RJ-Pa on day 18. We found that there were no significant differences in the ratios of FA(18:0), FA(18:1), FA(18:2), or FA(18:3) between the two royal jelly samples (Figure 4). This suggested that different types of bee pollen did not affect levels of 18-carbon saturated and unsaturated FAs in royal jelly, which might be related to lipid metabolism and regulation of maxillary glands during royal jelly secretion.

**Figure 2.** Changes in levels of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), and 10-hydroxydec-2-enoic acid (10-HDA) in royal jelly samples. Samples were collected on days 6, 12, 18, 24, and 30 after honeybees began feeding on single bee pollen. BP-Am, single bee pollen derived from *A. mono* Maxim.; BP-Pa, single bee pollen derived from *P. amurense* Rupr.; RJ-Am, royal jelly secreted by honeybees fed BP-Am; RJ-Pa, royal jelly secreted by honeybees fed BP-Pa. Significant differences in lipid content of RJ-Am or RJ-Pa secreted by honeybees fed BP-Am or BP-Pa at different timepoints are indicated with lowercase letters (a, b, c) and uppercase letters (A, B, C), respectively.

**Figure 3.** Changes in levels of 18-carbon saturated and unsaturated fatty acids in royal jelly secreted by honeybees fed single bee pollen derived from *Acer mono* Maxim. (BP-Am) or *Phellodendron amurense* Rupr. (BP-Pa). Samples were collected on days 18 and 24 after honeybees began feeding on single bee pollen. RJ-Am, royal jelly secreted by honeybees fed BP-Am; RJ-Pa, royal jelly secreted by honeybees fed BP-Pa. \*, *p*-value < 0.05.

**Figure 4.** The ratio of free and bound 18-carbon saturated and unsaturated fatty acids in royal jelly secreted by honeybees fed single bee pollen derived from *Acer mono* Maxim. (BP-Am) or *Phellodendron amurense* Rupr. (BP-Pa). RJ-Am, royal jelly secreted by honeybees fed BP-Am; RJ-Pa, royal jelly secreted by honeybees fed BP-Pa.
