Abstract
Geodia barretti is a deep-sea marine sponge common in the north Atlantic and waters outside of Norway and Sweden. The sampling and subsequent treatment as well as storage of sponges for metabolomics analyses can be performed in different ways, the most commonly used being freezing (directly upon collection or later) or by storage in solvent, commonly ethanol, followed by freeze-drying. In this study we therefore investigated different sampling protocols and their effects on the detected metabolite profiles in liquid chromatography-mass spectrometry (LC-MS) using an untargeted metabolomics approach. Sponges (G. barretti) were collected outside the Swedish west coast and pieces from three sponge specimens were either flash frozen in liquid nitrogen, frozen later after the collection cruise, stored in ethanol or stored in methanol. The storage solvents as well as the actual sponge pieces were analyzed, all samples were analyzed with hydrophilic interaction liquid chromatography as well as reversed phase liquid chromatography with high resolution mass spectrometry using full-scan in positive and negative ionization mode. The data were evaluated using multivariate data analysis. The highest metabolite intensities were found in the frozen samples (flash frozen and frozen after sampling cruise) as well as in the storage solvents (methanol and ethanol). Metabolites extracted from the sponge pieces that had been stored in solvent were found in very low intensity, since the majority of metabolites were extracted to the solvents to a high degree. The exception being larger peptides and some lipids. The lowest variation between replicates were found in the flash frozen samples. In conclusion, the preferred method for sampling of sponges for metabolomics was found to be immediate freezing in liquid nitrogen. However, freezing the sponge samples after some time proved to be a reliable method as well, albeit with higher variation between the replicates. The study highlights the importance of saving ethanol extracts after preservation of specimens for biology studies; these valuable extracts could be further used in studies of natural products, chemosystematics or metabolomics.
Highlights
IntroductionSponges (phylum Porifera) are benthic filter-feeding animals present in most marine and fresh-water habitats (van Soest et al, 2012)
Sponges are benthic filter-feeding animals present in most marine and fresh-water habitats
In total three specimens of the sponge G. barretti were collected in the Koster Sea outside the west coast of Sweden and from each specimen three technical replicates were stored in ethanol, methanol, frozen directly with liquid nitrogen or frozen back at the station after the collection cruise, respectively
Summary
Sponges (phylum Porifera) are benthic filter-feeding animals present in most marine and fresh-water habitats (van Soest et al, 2012). Geodia barretti (Geodiidae family, Tetractinellida order) is a massive deep-sea demosponge that is widely spread in the North Atlantic, including the Swedish West coast (Cárdenas et al, 2013; Cárdenas and Rapp, 2015) It is a key species of boreal sponge grounds Klitgaard and Tendal (2004), Murillo et al (2012), regularly studied for the many ecosystem services it provides Hoffmann et al (2009), Leys et al (2018), Maier et al (2020) and an important research organism in sponge biology Kutti et al (2015), Strand et al (2017), Koutsouveli et al (2020a), Koutsouveli et al (2020b), de Kluijver et al (2021), sponge cell culture Conkling et al (2019), sponge microbiology Radax et al (2012), Schöttner et al (2013), Luter et al (2017) as well as marine natural product research (Lidgren and Bohlin, 1986; Sjögren et al, 2004; Hedner et al, 2008; Carstens et al, 2015; Olsen et al, 2016a; Di et al, 2018)
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