Hairy marron (Cherax tenuimanus Smith) are critically endangered freshwater crayfish found only in a single river in south-west Australia. Conservation efforts have included a captive breeding program, which has been largely unsuccessful, despite the closely related smooth marron (Cherax cainii Austin) being successfully bred for aquaculture. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomic approach we created a profile of the metabolites in the haemolymph for males and females of the two species of marron. A non-lethal method was used to collect haemolymph and 84 reproducible annotated metabolites were identified. Variation in the levels of some metabolites were detected between species and between sexes within species. Multivariate analyses clearly differentiated the congeneric species and univariate analyses identified differences between species, sex and for some metabolite interactions between species and sex. This study created a baseline metabolome dataset for the two species and began to investigate the biological significance of metabolites that varied between species. We have shown metabolomics could be used for targeted studies to potentially assist reproductive success. This approach will be beneficial for conservation and aquaculture practices with potential applications for other aquatic taxa worldwide.

Captive breeding is a vital tool in the conservation of highly endangered species, as it is for the Margaret River hairy marron, Cherax tenuimanus, from the south west of Australia. A close relative, Cherax cainii, has almost completely displaced C. tenuimanus in the wild and is a successful aquaculture species, whereas C. tenuimanus has performed poorly in captivity. We used untargeted liquid chromatography-mass spectrometry to obtain metabolomic profiles of female and male C. tenuimanus held in controlled aquarium conditions during their reproductive period. Using repeated haemolymph sampling we tracked the metabolomic profiles of animals just prior to and for a period of up to 34 days after pairing with a similar sized potential mate. We identified 54 reproducible annotated metabolites including amino acids, fatty acids, biogenic amines, purine and pyrimidine metabolites and excretion metabolites. Hierarchical clustering analysis distinguished five metabolite clusters. Principal component-canonical variate analysis clearly distinguished females from males, both unpaired and paired; similar trends in profile changes in both sexes after pairing; and a striking shift in males upon pairing. We discuss three main patterns of metabolomic responses: differentiation between sexes; reactive responses to the disturbance of pairing; and convergent response to the disturbance of pairing for males. Females generally had higher concentrations of metabolites involved in metabolic rate, mobilisation of energy stores and stress. Responses to the disturbance of pairing were also related to elevated stress. Females were mobilising lipid stores to deposit yolk, whereas males had a rapid and strong response to pairing, with shifts in metabolites associated with gonad development and communication, indicating males could complete reproductive readiness only once paired with a female. The metabolomic profiles support a previously proposed potential mechanism for displacement of C. tenuimanus by C. cainii in the wild and identify several biomarkers for testing hypotheses regarding reproductive success using targeted metabolomics.