Interactions at the Oviposition Scar: Molecular and Metabolic Insights into Elaeagnus angustifolia's Resistance Response to Anoplophora glabripennis

Abstract

The Russian olive (Elaeagnus angustifolia), which functions as a "dead-end trap tree" for the Asian long-horned beetle (Anoplophora glabripennis) in mixed plantations, can successfully attract Asian long-horned beetles for oviposition and subsequently kill the eggs by gum. This study aimed to investigate gum secretion differences by comparing molecular and metabolic features across three conditions-an oviposition scar, a mechanical scar, and a healthy branch-using high-performance liquid chromatography and high-throughput RNA sequencing methods. Our findings indicated that the gum mass secreted by an oviposition scar was 1.65 times greater than that secreted by a mechanical scar. Significant differences in gene expression and metabolism were observed among the three comparison groups. A Kyoto Encyclopedia of Genes and Genomes annotation and enrichment analysis showed that an oviposition scar significantly affected starch and sucrose metabolism, leading to the discovery of 52 differentially expressed genes and 7 differentially accumulated metabolites. A network interaction analysis of differentially expressed metabolites and genes showed that EaSUS1, EaYfcE1, and EaPGM1 regulate sucrose, uridine diphosphate glucose, α-D-glucose-1P, and D-glucose-6P. Although the polysaccharide content in the OSs was 2.22 times higher than that in the MSs, the sucrose content was lower. The results indicated that the Asian long-horned beetle causes Russian olive sucrose degradation and D-glucose-6P formation. Therefore, we hypothesized that damage caused by the Asian long-horned beetle could enhance tree gum secretions through hydrolyzed sucrose and stimulate the Russian olive's specific immune response. Our study focused on the first pair of a dead-end trap tree and an invasive borer pest in forestry, potentially offering valuable insights into the ecological self-regulation of Asian long-horned beetle outbreaks.

Keywords: Asian long-horned beetle; Russian olive tree gum; dead-end trap tree; starch and sucrose metabolism.

Figure 1

The oviposition process of Asian long-horned beetles on Russian olives: (a) an adult beetle supplements nutrients on a twig; (b) adult beetles mate on a branch; (c) the female beetle uses her mouthparts to chew an oviposition scar; (d) subsequently, the female beetle rotates and uses an ovipositor to penetrate the scar for oviposition; (e) inside the scar, an egg encased in the transparent gum; (f) a comprehensive image of Russian olive.

Figure 1

The oviposition process of Asian long-horned beetles on Russian olives: (a) an adult beetle supplements nutrients on a twig; (b) adult beetles mate on a branch; (c) the female beetle uses her mouthparts to chew an oviposition scar; (d) subsequently, the female beetle rotates and uses an ovipositor to penetrate the scar for oviposition; (e) inside the scar, an egg encased in the transparent gum; (f) a comprehensive image of Russian olive.

Figure 2

Gum secreted from MSs and OSs: (a) weight of gum from MSs and OSs; (b) field images of the two adhesive treatments. Note: * indicates statistically significant differences (p < 0.05) as determined by the Mann–Whitney test. The red label denotes OSs, and the orange label denotes MSs.

Figure 3

DEGs in the transcriptomes of ALBs’ OSs, MSs, and HBs on Russian olive: (a) PCA plots; (b) illustration of significantly up- and down-regulated DEGs; (c) Venn diagram of common and specific DEGs.

Figure 4

Top 20 KEGG pathways in enrichment analysis of DEGs in OSs, MSs, and HBs of Russian olive: (a) OSs vs. HBs; (b) MSs vs. HBs; (c) OSs vs. MSs. The top 20 KEGG pathways are listed based on the Rich factor [the ratio of enriched genes in the pathway (sample number) to annotated genes (background number)], with the dot size representing the gene count and color indicating the p-value range. The branches of the KEGG metabolic pathways names are represented by different colors, which include metabolism (M), genetic information processing (GIP), environmental information processing (EIP), cellular processes (CPs), organismal systems (OSYs), human diseases (HDs), and drug development (DD).

Figure 4

Top 20 KEGG pathways in enrichment analysis of DEGs in OSs, MSs, and HBs of Russian olive: (a) OSs vs. HBs; (b) MSs vs. HBs; (c) OSs vs. MSs. The top 20 KEGG pathways are listed based on the Rich factor [the ratio of enriched genes in the pathway (sample number) to annotated genes (background number)], with the dot size representing the gene count and color indicating the p-value range. The branches of the KEGG metabolic pathways names are represented by different colors, which include metabolism (M), genetic information processing (GIP), environmental information processing (EIP), cellular processes (CPs), organismal systems (OSYs), human diseases (HDs), and drug development (DD).

Figure 5

Qualitative and quantitative analyses of the metabolome of OSs, MSs, and HBs: (a) number of metabolites in each category; (b) PCA of OSs, MSs, and HBs: (c) number of DAMs (|FC| ≥ 2) in OSs vs. HBs, MSs vs. HBs, OSs vs. MSs.

Figure 6

Top 20 lists of KEGG pathways in DAMs: (a) OSs vs. HBs; (b) MSs vs. HBs; (c) OSs vs. MSs. Note: The vertical axis represents the pathway name; the horizontal axis represents the Rich factor, the ratio of enriched genes in the pathway to annotated genes; and a higher Rich factor indicates greater enrichment. The dot size denotes gene count, and the dot color signifies p-value ranges.

Figure 7

The heatmap illustrates the saccharide content in OSs, MSs, and HBs. Different colors indicate the relative accumulation of DAMs, normalized using log2 fold change (log2FC). Red indicates the presence of the same major components of tree gum.

Figure 8

KEGG pathway enrichment analysis of DEGs and DAMs in the transcriptome and metabolome. Note: the horizontal axis represents metabolic pathways; the vertical axis represents the enriched p-values of DEGs (blue) and DAMs (orange) using −logP.

Figure 9

Gene expression patterns of DEGs between OSs and MSs: (a) heatmap of significant DEGs; (b) volcano plot of significant DEGs. Note: Blue indicates down-regulation and red indicates up-regulation. The vertical coordinate represents Padjust (significance level); the heatmap of gene expression is normalized in log2FC, and the clusters are defined based on Euclidean distance. (c,d) Gene expression analysis based on expression levels. Note: the number of genes in each cluster is indicated in brackets. Each line in the figure represents the change trend of a gene, and the blue line represents the change trend of the average expression level of all genes in the gene set.

Figure 10

Differentially enriched metabolites and genes in a glycolytic pathway: (a) OS vs. MS; (b) MS vs. HB; (c) OS vs. HB. Note: Boxes represent metabolites, solid lines and letters represent differentially enriched genes. Red indicates up-regulation and blue indicates down-regulation. Dashed lines and letters indicate that the metabolite or gene was not differentially enriched.

Figure 11

Integration analysis of DEGs and DAMs linked to the starch and sucrose metabolism pathway. The relative expression levels of DEGs and the accumulation of DAMs were calculated using log2FC.

Figure 12

Total sugar, total polysaccharide, sucrose, and glucose content in OSs, MSs, and HBs: (a) total sugar content; (b) total polysaccharide content; (c) sucrose content; (d) glucose content. Note: asterisks denote significant differences from control HBs according to Tukey’s multiple comparison test (n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 13

Proposed model for the response of Russian olive trees to oviposition scar caused by Asian long-horned beetles. In damage-associated molecular patterns (DAMPs), sucrose is unloaded from the phloem. (1) Sucrose is hydrolyzed by invertase (INV) into glucose, which is then converted by hexokinase (HXK) to glucose-6-phosphate (G6P). (2) Sucrose, uridine diphosphate glucose (UDPG), and D-glucose-1-phosphate (G1P) are mutually transformed by sucrose synthase (SUS), phosphodiesterase (YfcE), and small ribosomal subunit protein (RT02). In herbivore-associated molecular patterns (HAMPs), additional sucrose is degraded by SUS to UDPG, while the conversion of UDPG to G1P is inhibited by YfcE. More G6P is derived from G1P through phosphoglucomutase (PGM).