Production of methyl farnesoate by the mandibular organs of the mud crab, Scylla serrata: Validation of a radiochemical assay

Abstract

Parameters for the validation of a radiochemical assay to monitor the biosynthesis and release of methyl farnesoate (MF) by the mandibular organs (MO) of the mud crab, Scylla serrata, have been defined. On the basis of HPLC analysis, MF appeared to be the exclusive radiolabeled product of release, using [ 3H- methyl]methionine as precursor. HPLC of isooctane extracts of medium in which MO had been maintained similarly revealed that MF was the exclusive 3H-product and indicates that the isooctane partition assay can be employed to monitor MF release. The time course of MF biosynthesis and release suggested that the endogenous l-methionine pool is large, because both cumulative release and rate of release increased over an 8-hr incubation but remained constant between 8 and 16 hr. Glandular content of MF did not appear to stabilize until 6–8 hr after the start of the incubation. Cumulative MF release, as a function of glandular content or biosynthesis, increased gradually with time, but even after 16 hr the majority of biosynthesized MF was retained within the MO rather than released. l-Methionine concentration in the incubation medium influenced both MF biosynthesis and release, although the dose dependency was more apparent with biosynthesis. Although it was difficult to associate strictly a level of biosynthesis with l-methionine concentration, low rates of MF biosynthesis were observed at concentrations of less than 25 μ M. Because high levels of biosynthesis were observed consistently between 60 and 70 μ M l-met, all subsequent incubations employed a concentration of 65 μ M. MO of S. serrata show a striking asymmetry in MF biosynthesis and release which is not time dependent. Hence the comparison of right and left glands in assessing the effects of experimental treatments is precluded.