The lipid composition of the exo-metabolome from Haemonchus contortus
Authors: Godoy Pablo , Rezanezhad-Dizaji Behrouz , Buzatto Adriana Zardini , Sanchez Laura , Li Liang 
Published: January 2024

Background
Several livestock parasitic nematode species have developed anthelmintic resistance, limiting their control in grazing animals. Metabolomic studies of different parasite-derived biomolecules, such as lipids, are currently under investigation to expand novel strategies for controlling these pathogens and characterizing their metabolism. However, the extremely low concentrations and complexity of exocellular fluids produced by these organisms remain a challenge for untargeted approaches. Lipids are essential pleiotropic biomolecules in invertebrates, including helminths. Still, there is a gap in the knowledge about their functions in parasitic nematode biology and their impact on the interaction with the host. We aimed to describe the detailed lipid composition of the exo-metabolome secreted by Haemonchus contortus, the major parasitic nematode of small ruminants worldwide, using untargeted lipidomics.
Methods
H. contortus adult worms were recovered from infected sheep and cultured ex-vivo. Parasite medium was collected at three time points (2, 4 and 8h), and samples were subjected to an untargeted global lipidomic analysis. Firstly, lipids were isolated using a modified version of the classic Folch liquid-liquid extraction. The extracts were subjected to Liquid Chromatography-Mass Spectrometry (LC/MS) analysis. Chromatograms from samples and quality control injections were analyzed in positive and negative ionization. Lipid identification was run through a three-tier approach. For tiers 1 and 2, the experimental data was matched to databases through tandem-MS (MS/MS) spectral similarity scores. Lipids with MS/MS scores ≥ 500 were deemed Tier 1, while lower scores were considered Tier 2. Features not matched by MS/MS were subjected to mass-matching using the LIPID MAPS database (Tier 3). Lipids were subjected to a 9-tier filtering and scoring approach to select the best identification. The peak intensities were normalized by a set of internal standards to reduce experimental variability. The normalized peak intensities for identified lipids were subjected to statistical analysis comparing global lipid clusters' fold change (FC) and individual lipid features. Lipids were also analyzed by fatty acyl composition, segregating each lipid category by structural length and saturation bonding.
Results
Initially, 2562 lipid features were detected in all samples from our global untargeted lipidomic analysis, including background hits on the RPMI medium. A total of 1057 lipids were identified, including 171 lipids in Tiers 1 and 2 (high-confidence MS/MS identifications), whereas 886 putative lipids were identified in Tier 3 based on mass matches. The main lipid categories correspond to glycerophospholipids, followed by fatty acyls, sphingolipids, glycerolipids, and sterol lipids, structurally corresponding mainly to unsaturated lipids. We found variations in the parasite media lipid profile in a time-dependent manner. Many lipids have been described in multicellular organisms, but little is known about their role in parasitic nematodes. We predict their activity as signalling molecules in physiological functions such as adaptation to nutrient changes, life span and mating. Also, many lipids in the found profile have been documented as modulators on the host immune responses.
Conclusions
Our findings regarding the composition of the lipids secreted by H. contortus provide new information about how these biomolecules derived from this economically important veterinary nematode impact the host and how lipids play essential functions for the parasite’s homeostasis.