The smell of moulting:N-acetylglucosamino-1,5-lactone is a premoult biomarker and candidate component of the courtship pheromone in the urine of the blue crab,Callinectes sapidus

Abstract

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. 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. metabolites that can be used as an indicator of some biological state or condition) of pubertal premoult females. We tested this hypothesis by combining bioassay-guided fractionation and biomarker targeting. 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. The <500 Da fraction and the 500-1000 Da fraction but not the >1000 Da fraction of female urine induced male courtship stationary paddling, but none of the fractions of male urine did. Thus, female urine contains molecules of <1000 Da that stimulate courtship behaviours in males. 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. Purification, nuclear magnetic resonance, mass spectrometry and high pressure liquid chromatography analysis of this premoult biomarker identified it as N-acetylglucosamino-1,5-lactone (NAGL) and showed that it is more abundant in urine of premoult females and males than in urine of either postmoult or juvenile females and males. NAGL has not been reported before as a natural product or as a molecule of the chitin metabolic pathway. Physiological and behavioural experiments demonstrated that blue crabs can detect NAGL through their olfactory pathway. Thus, we hypothesize that NAGL is a component of the sex pheromone and that it acts in conjunction with other yet unidentified components.

Biomolecular changes that occur in the antennal gland of the giant freshwater prawn (Machrobrachium rosenbergii)

“…UHPLC-QToF-MS has received much attention in recent years for environmental metabolomics fingerprinting as well as in many other fields of chemical biology [12,43,44]. To extract the maximum number of metabolites within the AnG, a non-targeted extraction method was used.…”

“…Blue crabs in their pubertal molt stage release a sex pheromone in their urine, causing males to respond with courtship behaviour [12]. We have found the small molecule, NAGL (present in chitin biosynthesis pathway), a degradation product from the breakdown of chitin, in postmolt M .…”

“…Most of the associated biosynthesis pathway enzyme genes could be identified from AnG transcriptome data (Fig 6). NAGL has previously been identified as a pheromone in urine obtained from premolt female and male blue crabs [12], demonstrating that NAGL is more abundant in premolt female and male urine than in postmolt or juveniles. NAGL (logP = -3.04 (ACD/Labs Percepta Platform–PhysChem module for compound N -((3 R ,4 R ,5 S ,6 R )-4,5-dihydroxy-6-(hydroxymethyl)-2-oxotetrahydro-2 H -pyran-3-yl)acetamide) is more polar than NAG (logP = -2.48 ( N -((2 S ,3 R ,4 R ,5 S ,6 R )-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2 H -pyran-3-yl)acetamide) and would thus be more water soluble, ensuring a more pronounced transmission.…”

“…rosenbergii AnG transcriptome. Pathway figure modified from [12,56]. Biosynthetic enzymes-derived from M .…”

“…The most compelling evidence of a sex pheromone has been demonstrated from research on the blue crab, Callinectes sapidus , where N -acetylglucosamino-1,5-lactone (NAGL) present in the female AnG urine at intermolt and premolt, can activate physiological and behavioural responses in conspecifics [12]. Supporting a key role for the AnG in sex pheromone release, a recent transcriptomic screen of several tissues from the Eastern spiny lobster ( Sagmariasus verreauxi ), found that the most significantly biased transcript expression between males and females is within the AnG (other than the gonad) [13].…”