Metabolomic profiling of crayfish haemolymph distinguishes sister species and sex: implications for conservation, aquaculture and physiological studies

Abstract

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.

Detecting Sex-Related Changes to the Metabolome of a Critically Endangered Freshwater Crayfish During the Mating Season

“…Haemolymph was collected from the ventral sinus at the base of the 5th pereiopod using a 21G needle (Leland and Furse, 2012) and processed for LC-MS as described in Lette et al (2020). At the start of the run, a solvent blank, matrix blank and ten condition quality control samples consisting of a pooled quality control were analysed, with additional quality control samples injected after every fifth experimental sample following standard metabolomics protocols (Broadhurst et al, 2018).…”

“…Samples were analysed on a Ultra High-Pressure Liquid Chromatography pump (Dionex UltiMate 3000 RS) coupled to an Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific) fitted with a heated electrospray ionization probe (HESI) using a HPLC Hypersil, GOLD C18 (100 × 2.1 mm, 1.9 µM particle size column, Thermo Fisher Scientific) with an in-line filter. Details can be found in Lette et al (2020).…”

“…Metabolites were annotated according to Lette et al (2020) using an in-house MS/MS spectral database and the mzCloud online spectral library 1 and scored according to the Metabolomics Standards Initiative protocol (Sumner et al, 2007). Pathway enrichment followed the reference metabolome from Metaboanalyst 4.0 (Chong et al, 2019).…”

“…All identified metabolites were combined into a single data matrix and the multivariate covariance analysed using PCA (Jolliffe, 2002). Hierarchical cluster analysis (HCA) (Kaufman and Rousseeuw, 1990;Hastie et al, 2009) was then performed to assess the similarities between individual metabolite concentrations, both PCA and HCA as described in Lette et al (2020). For this study, a Principal Component-Canonical Variate Analysis (PC-CVA) was conducted using a five principal components projection to illustrate multivariate discrimination between the clusters.…”

“…Whilst still new to the aquaculture industry and captive breeding of aquatic organisms (Alfaro and Young, 2016), metabolomics has been used to assess the health of stock by identifying biomarkers for stressors to environmental conditions for aquatic organisms, including decapod crustaceans, such as oxidative stress in Callinectes sapidus (Schock et al, 2010), Procambarus clarkii (Izral et al, 2018); oxidative and thermal stress in Crassula aequilatera (Alfaro et al, 2019); nutritional stress in P. clarkii (Izral et al, 2018), Astacus leptodactylus (Costantini et al, 2018); anthropogenic stressors such as metals and contaminants in Orconectes virilis (Izral, 2016); and overall health in high density stocking of Litopenaeus vannamei (Schock et al, 2013). Metabolomic profiles for unpaired C. cainii and C. tenuimanus have been created using untargeted liquid chromatography-mass spectrometry (LC-MS) of haemolymph (Lette et al, 2020). The two species were in different reproductive and growth stages at the time of the study; C. cainii had completed breeding and moulting was about to commence, whereas C. tenuimanus were still mating.…”