Differential Metabolic Profiles during the Developmental Stages of Plant-Parasitic Nematode Meloidogyne incognita

Abstract

Meloidogyne incognita is a common root-knot nematode with a wide range of plant hosts. We aimed to study the metabolites produced at each stage of the nematode life cycle to understand its development. Metabolites of Meloidogyne incognita were extracted at egg, J2, J3, J4, and female stages and 110 metabolites with available standards were quantified using CE-TOF/MS. Analyses indicated abundance of stage-specific metabolites with the exception of J3 and J4 stages which shared similar metabolic profiles. The egg stage showed increased abundance in glycolysis and energy metabolism related metabolites while the J2 metabolites are associated with tissue formation, motility, and neurotransmission. The J3 and J4 stages indicated amino acid metabolism and urea cycle- related metabolites. The female stage was characterized with polyamine synthesis, antioxidant activity, and synthesis of reproduction related metabolites. Such metabolic profiling helps us understand the dynamic physiological changes related to each developmental stage of the root-knot nematode life cycle.

Keywords: Meloidogyne incognita; developmental stages; metabolic pathways; metabolic profiles; root-knot nematode.

Figure 1

(A) Diversity of the metabolites observed at each stage of the nematode life cycle indicating major metabolic pathways of the cellular metabolism to which they constitute; (B) PCA loading plot of the two first principal components of the metabolites of M. incognita at various stages. The sum of two components amounted to 77.8%. Here, the metabolites can be observed to be grouped based on the stages where their expression has been significantly observed. The colored dots indicate the stage at which the particular metabolite showed highest quantification.

Figure 2

Heat map comparing the levels of 93 metabolites detected in Meloidogyne incognita at various stages of its life cycle. For construction of a comparative heatmap absolute quantified values were normalized and relative expression represented at a range of −1.67 to 1.93 was used. The metabolites were grouped based on their metabolism into amino acid metabolism, BCAA and aromatic amino acid metabolism, central carbon metabolism, coenzyme metabolism, lipid metabolism, nucleotide metabolism, urea cycle-related metabolism and others. Generation of the heatmap was carried out using MeV (v4.9.0) software [15]. Below: Pathway maps indicating the expression of its constituent metabolites. G1P, glucose 1 phosphate; G6P, glucose 6 phosphate; F6P, fructose 6 phosphate; F1,6P, fructose 1,6 phosphate; DHAP, dihydroxyacetone phosphate; 3-PG, 3-phosphoglycerate; G3P, glyceraldehyde 3 phosphate; 2-PG, 2-phosphoglycerate; PEP, phosphoenol pyruvate; 3-HBA, 3-hydroxybutyrate; b-Ala, beta alanine; BTL, betaine aldehyde; DMG, N,N-Dimethylglycine.

Figure 3

Morphology of the root-knot nematode Meloidogyne incognita at different stages of development and stage-specific highly expressed metabolites at each stage of the life cycle; ATP, adenosine triphosphate; NAD⁺, nicotinamide adenine dinucleotide. All metabolites concentrations were calculated by normalizing the peak area of each metabolite samples with respect to the area of the internal standard and by using standard curves, which were obtained by single-point (100 µM) calibrations. Scale bars in the representative stage images are egg (0.2 mm), J2 (0.4 mm), J3 (0.4 mm), J4 (0.4 mm) and female (1.0 mm).