Estimation of lipid reserves in different life stages of Meloidogyne incognita using image analysis of Nile Red-stained nematodes

Abstract

Summary Biochemical analyses of nematodes have revealed that neutral lipids (especially triglycerides) are the main source of energy reserves, which is depleted as the nematodes age. Several methodologies have been developed to visualise triglyceride-rich fat stores in plant-parasitic nematodes using non-fluorescent, lipophilic dyes, such as Oil Red O. Here, we propose a robust and reproducible fluorescence-based Nile Red staining method (followed by image analysis) for rapid detection of neutral lipid droplets in Meloidogyne incognita. This unique lipophilic dye selectively fluoresces in red and green spectra in a lipid-rich environment. The neutral lipid content of M. incognita juveniles gradually diminished during different periods of food deprivation, and this was significantly correlated with reduction in parasitic success of M. incognita in eggplant. Additionally, variation in fat reserves in different developmental stages of M. incognita infecting adzuki bean was also demonstrated. This investigation may aid future metabolic research, including functional analysis of lipid regulatory genes in plant-parasitic nematodes.