Since scav-5 and scav-6 are paralogs of scav-4, we analysed their functions in lipid accumulation using scav-5(ok1606) deletion mutants and scav-6 knockout alleles generated in this study through CRISPR/Cas9-mediated gene editing (Figure 4B). We found that when fed with JUb74, both scav-5(-) and scav-6(-) mutants had moderately reduced LD sizes, but not to the extent of scav-4(-) mutants (Figure 4E). Previous promoter reporter studies showed that scav-5 and scav-6 were expressed in the intestine.34 We constructed translational reporters for both genes and found weak or no signals for SCAV-5::TagRFP possibly due to low protein levels. The SCAV-6::TagRFP fusion protein was expressed in the intestine and was localized to the apical membrane (Figure 4C). From the fluorescent intensity, the scav-6 expression appeared to be weaker than the scav-4 expression. Moreover, scav-4(-) scav-6(-) double mutants had the same LD diameter as scav-4(-) single mutants (Figure 4F). The above results suggested that SCAV-4 may play a more significant role than the other two paralogs in intestinal lipid uptake.

In a small-scale screen of a few thousand haploid genomes, we isolated an allele unk28, which led to the formation of supersized LDs under the JUb74 diet (Figure 4A).

Conversely, we also supplemented either specific BCFAs or a mixture of them to the OP50 culture but were not able to increase the BCFA levels above 30%, likely because OP50 could not take in large amount of BCFAs (Figure S2C). Feeding those BCFA-treated OP50 to wild-type C. elegans did not induce large LDs (Figure S2D). Thus, we concluded that, in addition to the dosage effect, a threshold existed for the minimal level of BCFAs that could induce large LDs.