Epistasis analysis suggests that trd-1 functions downstream of tra-2 and upstream of fem-3.

Worms were bleach synchronized at 15°C then L1s hatched, fed and shifted to 20°C or 25°C as indicated. Following whole worm DAPI on L4+1 day old animals, the presence of sperm (Sp) and oocytes (Oo) was recorded. fog-1(q253, lf) and fem-3(e2006, lf) mutants produce only oocytes at the restrictive temperature of 25°C, a phenotype which is unaltered by trd-1 RNAi, suggesting that trd-1 is operating upstream. The fbf-2(q738) mutation has a 23% penetrant feminized germline phenotype at 20°C which is completely suppressed by trd-1 RNAi. These animals are masculinized to the same extent as trd-1 RNAi animals alone, suggesting that trd-1 operates downstream of (or in parallel to) fbf-2. tra-2 (e2020, gf) mutants are feminized at 20°C, a phenotype that is significantly suppressed by trd-1 RNAi (again, these animals are masculinized to a similar extent to trd-1 RNAi animals alone). This suggests that trd-1 is downstream of tra-2. Overall, therefore, trd-1 appears to be operating downstream of tra-2 but upstream of fem-3 to regulate germline sex determination.Epistasis analysis suggests that trd-1 functions downstream of tra-2 and upstream of fem-3.