Description

The FLP family of neuropeptides contains at least 30 genes in C. elegans (Li and Kim, 2008). Some of the flp genes were found to play roles in locomotion, egg laying, and sleep, but most flp genes have unknown function (Li and Kim, 2008; Chang et al. 2015). flp-1 is one of the few flp genes whose mutations are reported to cause overt behavioral defects (Nelson et al. 1998; Hums et al. 2016; Buntschuh et al. 2018; Oranth et al. 2018). The initial flp-1 deletion alleles that were analyzed behaviorally (yn2 and yn4; Nelson et al. 1998) included a deletion of part of daf-10 (Ailion and Thomas, 2003; Buntschuh et al. 2018). This resulted in some of the initial behaviors that were attributed to FLP-1 peptides being caused by loss of daf-10 activity (Buntschuh et al. 2018). Subsequent analysis of newer flp-1 deletion alleles suggests that FLP-1 peptides are involved in nose touch sensitivity, locomotion, and reproduction (Buntschuh et al. 2018).

flp-1(ok2811) is a prospective null allele (Hums et al., 2016; Buntschuh et al.. 2018). We obtained strain RB2126 (Caenorhabditis Genetics Center, CGC) in order to analyze flp-1(ok2811) null mutant locomotion behavior. When the strain was grown at room temperature (23°C), some animals formed noticeable dauers similar to strains containing mutations in constitutive dauer (Daf-c) genes (Hu, 2007) (Fig. 1A). To examine whether the Daf-c phenotype was because of a background mutation, strain RB2126 was outcrossed three times. This outcross resulted in the segregation of two different mutants with readily apparent phenotypes: one resulting in deep body bends and a second in the Daf-c phenotype. PCR genotyping showed that the deep body bends co-segregated with the flp-1(ok2811) deletion. The mutation causing the Daf-c phenotype was mapped to chromosome V close to the daf-11 gene and a complementation test using daf-11(sa195) (a presumed null allele of daf-11) showed that it is in daf-11 (Birnby et al. 2000). These results suggest that flp-1(ok2811) mutants exhibit elevated body curvature, and that strain RB2126 has a background mutation in daf-11. This new daf-11 allele in strain RB2126 was named yak155.

flp-1(ok2811) mutants are known to exhibit elevated body curvature (Nelson et al. 1998; Chang et al. 2015; Hums et al. 2016, Buntschuh et al. 2018). Interestingly, strain RB2126 (flp-1(ok2811); daf-11(yak155)) had significantly reduced deep body bends in comparison to strain XZ2314 (flp-1(ok2811); three times outcrossed from strain RB2126) (Fig. 1B, 1D). This suggests that either the daf-11(yak155) mutation partially suppresses the phenotype of flp-1(ok2811) or that the RB2126 strain has other background mutation(s) that affect the body curvature. To test the possibility that the reduced deep body bends of flp-1(ok2811) in RB2126 strain are due to daf-11, double mutant strain flp-1(ok2811); daf-11(sa195) was constructed, and its body curvature was examined. flp-1(ok2811); daf-11(sa195) mutants (strain XZ2380) had similar body curvature as flp-1(ok2811) mutants (strain XZ2314) (Fig. 1B-1E). In addition, larvae (L2 to L3 stage) of flp-1(ok2811) and flp-1(ok2811); daf-11(sa195) often exhibit a characteristic body posture resembling the number ”8”, but this posture was not observed in RB2126 larvae (Fig. 1C). This result shows that daf-11(sa195) does not affect the elevated body curvature of flp-1(ok2811) mutants, suggesting that the body curvature of the RB2126 strain may be modified by another background mutation(s) other than daf-11(yak155).

To verify the presence of additional background mutation(s), the body curvature of flp-1(ok2811); daf-11(yak155) double mutants (strain XZ2406) and flp-1(ok2811) mutants (strain XZ2314) were compared. Body curvature in these strains was found to be similar, suggesting that strain RB2126 has at least one additional background mutation in an unknown gene that affects the body curvature of flp-1(ok2811) (Fig. 1C, 1F). In support of this, strain RB2126 was outcrossed using strain EG7765 that contains the genetic marker oxTi76 (oxTi76 contains a transposon insertion of Peft-3::GFP::H2B::tbb-2 3’UTR on chromosome VI, in a location close to the flp-1 gene) and animals of the F2 generation that were not green, and did not exhibit Daf-c or elevated body curvature were picked. This outcross resulted in the isolation of strain XZ2394 that contains the flp-1(ok2811) mutation but does not show the same extent of elevated body curvature as XZ2314 (flp-1(ok2811)) strain (Fig. 1G). This suggests that strain RB2126, besides the mutation in daf-11, contains an additional mutation that partially but significantly suppresses the flp-1(ok2811) elevated body posture. This new allele was named yak156 and the updated genotype of strain RB2126 is flp-1(ok2811); daf-11(yak155); yak156.