Biomarker targeting using nuclear magnetic resonance (NMR) spectral analysis of the 500–1000 Da fraction of urine from premoult and postmoult males and females revealed a premoult biomarker. Thus, female urine contains molecules of <1000 Da that stimulate courtship behaviours in males. The 1000 Da fraction of female urine induced male courtship stationary paddling, but none of the fractions of male urine did. To evaluate the molecular mass of the putative pheromone by bioassay-guided fractionation, we separated urine from pubertal premoult females and intermoult males by ultrafiltration into three molecular mass fractions. We tested this hypothesis by combining bioassay-guided fractionation and biomarker targeting. metabolites that can be used as an indicator of some biological state or condition) of pubertal premoult females. We hypothesized that pheromones are mixtures of molecules and are more concentrated in urine of pubertal premoult females compared with other moulting stages and thus that these molecules are biomarkers (i.e. Female blue crabs ( Callinectes sapidus) in their pubertal moult stage release unidentified sex pheromone molecules in their urine, causing males to respond with courtship behaviours including a display called courtship stationary paddling and a form of precopulatory guarding called cradle carry.
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