2.5. Biochemical assays
Briefly, nematodes were homogenized followed by a centrifugation at 4 ÂșC, and the supernatants were aliquoted for subsequent assays. Regarding the neural signals, the contents of 5-HT, DA, ACh and GABA and the activities of AChE were determined using the commercial enzyme-linked immune-sorbent assay (ELISA) kits (Shanghai Ketao Biotechnology Co. LTD) according to the instructions (Liu et al., 2020; Yue et al., 2021). Regarding the lipid metabolism, the contents of triacylglycerol (TG), non-esterified fatty acid (NEFA) and the metabolites acetyl-CoA (ACA) and acyl-CoA (FA-CoA) that combine both lipogenesis and lipolysis were measured. In addition, the activities of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and glycerol-3-phosphateacyl transferases (GPAT), which facilitate lipogenesis, and lipase, acyl-CoA synthetase (ACS) and carnitine palmitoyl transferase (CPT), were analyzed. Finally, adipose triglyceride lipase (ATGL) that facilitates lipolysis was also measured. Measurements were conducted with the ELISA method (Zhang et al., 2022). Briefly, the ELISA kits provided 96-well plates, substrates, buffer and standard enzyme solutions. Sufficient substrates were added with a series of enzyme solutions (diluted with the buffer) whose concentrations were known, and the results were used to establish standard curves which were used to calculate the results of the samples. In each group, three were at least six replicates with 500 nematodes in each to ensure the methodological accuracy (Zhang et al., 2017). The biomass among samples was balanced by the normalization where each biochemical was represented as its proportion against the total protein (TP) in each sample. Each group was composed of at least three replicates.