(A–C) Nereidin activity elutes in lipophilic fractions of head extracts but is not proteinaceous as previously hypothesised. (A) Schematised fractionation of premature Platynereis head extracts, expected to recover the described nereidin activity in the methanolic eluate of SPE. (B) Relative coelomocyte vtg levels after overnight incubation with flowthrough or eluate of (a), normalised against control samples. (C) Insensitivity of nereidin activity against proteinase and heat treatment. Experiment as in (b), with pretreatment of eluate by proteinase K (‘ProtK’) and/or subsequent heat treatment (5’, 95°C; ‘Heat’). Results indicate that the repressive effect of nereidin on eleocyte vtg levels is neither sensitive to proteinase nor heat. (D–G) Identification of methylfarnesoate as a major component of nereidin. (D) Further fractioning of the brain hormone activity (eluate from b) by C18-reverse phase column chromatography. Absorption of eluent at 280 nm (red line) and 215 nm (black line) during application of acetonitrile gradient (dashed line, right ordinate). Peak 15 (marked red), containing the nereidin activity, elutes at a retention time of 20.5 min. (E) Peak 15, containing methylfarnesoate (MF), significantly suppresses vtg levels in the coelomocyte bioassay when compared to the controls. (F) Authentic MF recapitulates the observed effect at physiological concentration ranges; relative vtg levels in coelomocytes treated with 0.1 nM – 1000 nM MF in 0.01% DMSO, normalised to the control group of DMSO-treated cells. (G) Specificity of the repressive effect caused by MF; comparison of coelomocyte vtg levels after incubation in 10 nM MF, palmitic acid (PA) or retinoic acid (RA). Graphs in (B,C,E,F,G) show qRT-PCR quantification of vtg levels after overnight (20 hr) treatment of primary coelomocyte cultures. Expressions values were calculated with respect to the reference gene rps9, and normalised to the levels of controls (untreated cells). Boxplots show the first and third quartile, the median (solid line), and the mean (dashed line). Whiskers denote the 10th and 90th percentile. Statistical significance was tested by a one-sided t-test of the treatment group against the control, or, in case of multiple comparisons, first with an ANOVA, followed by a one-sided t-test with adjustment of the p-values for multiple testing to assess differences between the different groups. *p<0.05. **p<0.01, ***p<0.001. n: number of biological replicates. Raw data for panels B,C,E,F,G provided in Figure 2—source data 1.