Protocol for metabolomics sampling of bivalve haemolymph and tissues Thao V. Nguyen, Andrea C. Alfaro and Tim Young Aquaculture Biotechnology Research Group, Auckland University of Technology, Email:
[email protected]
Aims The protocol aims to describe step by step the process of sampling and metabolism quenching of bivalve plasma and tissues that used for metabolomics purpose.
Materials Biological materials: bivalve species. Reagents and reagent setup • • •
Ethanol 70% (for disinfection). Liquid nitrogen (for metabolism quenching). Autoclaved artificial salt water (ASW). Preparation of autoclaved-ASW: Please see Nguyen (2018).
Equipment • • • •
Needle (23 gauge x 1.5”) attached to a 3 mL sterile syringe (Terumo, Japan). Dissection tools. 1.5 mL Eppendorf tubes. 2 mL BioStorTM cryovials (National Scientific Supply Company, Carlifornia, USA).
Methods Method described here is based on the published protocol (Nguyen et al. 2018d). Steps 1. Disinfect the working table. 2. Take the animals out of water and clean outside with tissue paper. 3. Gently open animal valves and pour out all water inside the animal. 4. Gently insert needle into posterior adductor muscle of the animal and withdraw approximately 1 mL haemolymph (0.6 for metabolomics and 0.4 for other purposes). 5. Transfer the haemolymph into 1.5 mL vial placed on ice. 6. Transfer 0.6 mL of haemolymph into 2 mL cryovial and immediately freeze in liquid nitrogen. NOTE: For flow cytometric analyses with Muse Cell Analyser (Merck Millipore) (Nguyen et al. 2018a), haemolymph samples can be maintained by mixing with cold autoclaved-ASW (1:1) and incubate on ice prior analyses. 7. Dissect the animal, cut approximately 1 g of each tissue (gills, haepatopancrease and others of interest) and then transfer each tissue individually into 2 mL cryovial and immediately freeze in liquid nitrogen. NOTE: If multiple tissues are processed, disinfect the dissection tools (knife, scissor…) in 70% ethanol and washed with autoclaved-ASW before continue with the next tissue. 8. Clean and disinfect the dissection table and tools before moving to the next animal. 9. At the end of the sampling, take all vials out of liquid nitrogen and store at -80°C until they could be analysed (Nguyen et al. 2018b; c).
1
References: Nguyen, T. V. 2018. Preparation of Artificial Sea Water (ASW) for Culturing Marine Bacteria. Nguyen, T. V., Alfaro, A. C., Merien, F., Young, T. & Grandiosa, R. 2018a. Metabolic and immunological responses of male and female New Zealand Greenshell™ mussels (Perna canaliculus) during Vibrio sp. infection. Journal of Invertebrate Pathology. Nguyen, T. V., Alfaro, A. C. & Young, T. 2018b. Protocol for Metabolite Extraction from Marine Bivalve Haemolymph. Nguyen, T. V., Alfaro, A. C. & Young, T. 2018c. Protocol for Methyl ChloroFormate (MCF) Derivatisation of Extracted Metabolites from Marine Bivalve Tissues. Nguyen, T. V., Alfaro, A. C., Young, T., Ravi, S. & Merien, F. 2018d. Metabolomics Study of Immune Responses of New Zealand Greenshell™ Mussels (Perna canaliculus) Infected with Pathogenic Vibrio sp. Marine Biotechnology, 114.
2
