Workflow for the Targeted and Untargeted Detection of Small Metabolites in Fish Skin Mucus

Lada Ivanova,Søren Grove,Haitham Tartor,Silvio Uhlig,Anja Kristoffersen

doi:10.3390/fishes3020021

Abstract

The skin mucus of fish is in permanent contact with the aquatic environment. Data from the analysis of the chemical composition of skin mucus could potentially be used for monitoring the health status of the fish. Knowledge about mucus composition or change in composition over time could also contribute to understanding the aetiology of certain diseases. The objective of the present study was the development of a workflow for non-invasive sampling of skin mucus from farmed salmon (Salmo salar) for the targeted and untargeted detection of small metabolites. Skin mucus was either scraped off, wiped off using medical wipes, or the mucus’ water phase was absorbed using the same type of medical wipes that was used for the wiping method. Following a simple filtration step, the obtained mucus samples were subjected to hydrophilic interaction chromatography coupled to high-resolution mass spectrometry. Post-acquisition processing included the targeted analysis of 86 small metabolites, of which up to 60 were detected in absorbed mucus. Untargeted analysis of the mucus samples from equally treated salmon revealed that the total variation of the metabolome was lowest in absorbed mucus and highest in the scraped mucus. Thus, future studies including small-molecule metabolomics of skin mucus in fish would benefit from a sampling regime employing absorption of the water phase in order to minimize the bias related to the sampling step. Furthermore, the absorption method is also a less invasive approach allowing for repetitive sampling within short time intervals.

Highlights

An increasing world population size, standard of living, and globalization have placed excessive demands on the aquaculture industry that according to the Food and Agriculture Organization of the United Nations needs to double food production by mid-century [1]
An antibacterial effect of fish skin mucus was reported in several species [8,9]
The selection of the 86 metabolite standards for the targeted analysis of fish skin mucus was based on a recent metabolite profiling study [10]

Summary

Introduction

An increasing world population size, standard of living, and globalization have placed excessive demands on the aquaculture industry that according to the Food and Agriculture Organization of the United Nations needs to double food production by mid-century [1]. The economic success of the modern aquaculture is intimately attributed to the health and welfare of the aquatic animals [2]. In this context, mucus plays a central role in maintaining fish health providing a physical and biochemical barrier against a broad spectrum of pathogens present in aquatic environments [3]. Fish skin mucus was demonstrated to be a source for numerous immune-related components such as lysozymes, phosphatases, esterases, proteolytic enzymes, complement factors, lectins, immunoglobulins, C-reactive proteins, Fishes 2018, 3, 21; doi:10.3390/fishes3020021 www.mdpi.com/journal/fishes. An antibacterial effect of fish skin mucus was reported in several species [8,9]. The observed antibacterial activity was stronger toward Gram positive compared to Gram negative bacteria, but the key bioactive components have not yet been identified

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