Metabolic Analysis of the Development of the Plant-Parasitic Cyst Nematodes Heterodera schachtii and Heterodera trifolii by Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Abstract

The cyst nematodes Heterodera schachtii and Heterodera trifolii, whose major hosts are sugar beet and clover, respectively, damage a broad range of plants, resulting in significant economic losses. Nematodes synthesize metabolites for organismal development and social communication. We performed metabolic profiling of H. schachtii and H. trifolii in the egg, juvenile 2 (J2), and female stages. In all, 392 peaks were analyzed by capillary electrophoresis time-of-flight mass spectrometry, which revealed a lot of similarities among metabolomes. Aromatic amino acid metabolism, carbohydrate metabolism, choline metabolism, methionine salvage pathway, glutamate metabolism, urea cycle, glycolysis, gluconeogenesis, coenzyme metabolism, purine metabolism, pyrimidine metabolism, and tricarboxylic acid (TCA) cycle for energy conversion (β-oxidation and branched-chain amino acid metabolism) energy storage were involved in all stages studied. The egg and female stages synthesized higher levels of metabolites compared to the J2 stage. The key metabolites detected were glycerol, guanosine, hydroxyproline, citric acid, phosphorylcholine, and the essential amino acids Phe, Leu, Ser, and Val. Metabolites, such as hydroxyproline, acetylcholine, serotonin, glutathione, and glutathione disulfide, which are associated with growth and reproduction, mobility, and neurotransmission, predominated in the J2 stage. Other metabolites, such as SAM, 3PSer, 3-ureidopropionic acid, CTP, UDP, UTP, 3-hydroxy-3-methylglutaric acid, 2-amino-2-(hydroxymethyl-1,3-propanediol, 2-hydroxy-4-methylvaleric acid, Gly Asp, glucuronic acid-3 + galacturonic acid-3 Ser-Glu, citrulline, and γ-Glu-Asn, were highly detected in the egg stage. Meanwhile, nicotinamide, 3-PG, F6P, Cys, ADP-Ribose, Ru5P, S7P, IMP, DAP, diethanolamine, p-Hydroxybenzoic acid, and γ-Glu-Arg_divalent were unique to the J2 stage. Formiminoglutamic acid, nicotinaminde riboside + XC0089, putrescine, thiamine 2,3-dihydroxybenzoic acid, 3-methyladenine, caffeic acid, ferulic acid, m-hydrobenzoic acid, o- and p-coumaric acid, and shikimic acid were specific to the female stage. Overall, highly similar identities and quantities of metabolites between the corresponding stages of the two species of nematode were observed. Our results will be a valuable resource for further studies of physiological changes related to the development of nematodes and nematode-plant interactions.

Keywords: CE-TOF/MS; Heterodera schachtii; Heterodera trifolii; cyst nematodes; metabolic pathways.

Figure 1

Integrated superimposed metabolic pathway map. Carbohydrate metabolism (1); purine metabolism (2,3); glycolysis and gluconeogenesis metabolism (4); pyrimidine metabolism (5); TCA cycle to store energy (6); TCA cycle for energy conversion: β-oxidation and BCAA metabolism (7); aromatic amino acid metabolism-phenylalanine and tyrosine (8); choline metabolism and methionine salvage (9); glutamate metabolism and urea cycle (10); and aromatic amino acid metabolism- tryptophan (11). The bars/lines represent peak areas of metabolites in H. schachtii egg (blue), H. schachtii juvenile 2 (red), H. schachtii female (green), H. trifolii egg (orange), H. trifolii juvenile 2 (mazarine), H. trifolii female (purple), respectively. Boxes with no bars indicate metabolites not detected. Refer Table S2 for the abbreviations.

Figure 2

Top fifty metabolites in the egg, juvenile 2 (J2), and female stages of H. schachtii and H. trifolii nematode species arranged in descending order (top to bottom) of relative peak area (mg⁻¹). Refer to Table S2 for abbreviations of metabolites. The unknown metabolites XA0033 (^a) (S/NO, 355; Table S2) and XC0089 (^b) (S/No, 365; Table S2) had predicted m/z values of 242.080 and 255.099, respectively.

Figure 3

Score plots of the principal component analysis (PCA) (top) and orthogonal partial least squares discriminant analysis (OPLS-DA) (bottom) of H. schachtii and H. trifolii based on the relative peak areas of metabolites. HS-E, H. schachtii egg stage; HS-J2, H. schachtii juvenile 2 stage; HS-F, H. schachtii female stage; HT-E, H. trifolii egg stage; HT-J2, H. trifolii juvenile 2 stage; and HT-F, H. trifolii female stage.

Figure 4

Heat map of changes in metabolite levels at the egg, J2, and female stages of H. schachtii (HS) and H. trifolii (HT). (A) metabolites involved in the metabolic pathways. (B) Metabolites not involved in the biosynthetic pathways. Comparative heat maps were constructed after quantile normalization of relative peak areas in the range of 0.0000 to 0.0.00082. Red indicates increased metabolite levels, green represents decreased levels, and black indicates intermediate levels. The heat map was generated using MeV (ver. 4.9.0) software. HS Egg, H. schachtii egg stage; HS J2, H. schachtii juvenile 2 stage; HS F, H. schachtii female stage; HT Egg, H. trifolii egg stage; HT J2, H. trifolii juvenile 2 stage; and HT female, H. trifolii female stage. Refer to Table S2 for abbreviations of metabolites.

Figure 5

Stage-specific metabolites in H. schachtii and H. trifolii with their functions and biosynthetic pathways. Photographs of nematodes in the egg, J2, and female stages (A); metabolites highly detected at only the egg stage (B); J2 stage (C); and female stage (D). Cellular locations and pathways as defined in HMDB.