Potential of MALDI−TOF MS-based proteomic fingerprinting for species identification of Cnidaria across classes, species, regions and developmental stages
Sven Rossel*1, Janna Peters2, Silke Laakmann3,4, Pedro Martínez Arbizu1& Sabine Holst2
1 Senckenberg am Meer, German Centre for Marine Biodiversity Research (DZMB), 26382 Wilhelmshaven, Germany
2 Senckenberg am Meer, German Centre for Marine Biodiversity Research (DZMB), 20146 Hamburg, Germany
3Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg (HIFMB), 26129 Oldenburg, Germany
4Alfred Wegener Institute, Helmholtz-Centre for Polar and Marine Research (AWI), 27570 Bremerhaven, Germany
Corresponding author email: sven.rossel@senckenberg.de
Abstract
Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) into our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analyzed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies.
Introduction
Cnidaria is a highly diverse phylum currently including six accepted classes (WoRMS; 2022). Two classes, Anthozoa and Staurozoa, comprise benthic species (Mills et al., 2007; Miranda et al., 2018). Three classes (Scyphozoa, Hydrozoa, Cubozoa) include species with metagenetic life cycles, usually with a sexually reproducing planktonic medusa generation and an asexually reproducing benthic polyp generation, however, there are many exceptions from these reproduction modes (Bouillon et al., 2006; Jarms and Morandini, 2019). Another class (Myxozoa) are endoparasitic species diverged from free-living cnidarian ancestors (Okamura et al., 2015). The free-living species of the first five classes are distributed in marine ecosystems across all depths and latitudes (Mills et al., 2007; Rodríguez et al., 2014; Miranda et al., 2018; Jarms and Morandini, 2019). However, distribution and ecology of cnidarians are largely unexplored and cnidarian species are often neglected in biodiversity studies because of difficulties in their correct species identification (Häussermann, 2004; Martell et al., 2022). In particular, juvenile stages often lack diagnostic characters making identification to the species level impossible in many cases (Holst, 2012; Schuchert, 2012). However, morphological species identification is challenging because of lacking distinct morphological features and high morphological variation in many cnidarians (Brugler et al., 2018; Pruski and Miglietta, 2019; Lawley et al., 2021). Moreover, fixation of the gelatinous medusae and the soft-bodied polyps can lead to shrinkage and deformation of fragile specimens leading to distortion of morphological diagnostic features (Häussermann, 2004; Schuchert, 2012; Holst and Laakmann, 2014; Holst et al., 2019).
Molecular genetic techniques have been widely tested and applied for identification and differentiation of cnidarian species (Holst and Laakmann, 2014; Moura et al., 2018; Holst et al., 2019; Bucklin et al., 2021). However, not in all groups, standard fragments such as the COI barcode regions are fully informative on species level (Brugler et al., 2018; Schuchert, 2020). In some taxa, a combination of several molecular genetic markers achieves a satisfying resolution (McFadden et al., 2011, 2014). As an alternative to the expensive and time-consuming molecular genetic analyses, proteomic fingerprinting was successfully used for species identification in Cnidaria (Holst et al., 2019; Park et al., 2021; Korfhage et al., 2022). Using matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry, a subset of the specimen’s peptides and proteins are measured resulting in a mass spectrum that can be used for differentiation of species. The method is already widely established for routine pathogen identification (Chen et al., 2021) and is more recently also used in pathogen-vector identification, of mainly insects and mites (Dieme et al., 2014; Lafri et al., 2016; Hamlili et al., 2021). The low costs and short hand-on times per specimen (Tran et al., 2015; Rossel et al., 2019) coupled with high identification success makes it a promising tool for rapid and reliable species identification also in biodiversity assessments. In marine science it was applied for identification of a variety of animal groups such as copepods (Laakmann et al., 2013; Bode et al., 2017; Kaiser et al., 2018; Rossel and Martínez Arbizu, 2019), isopods (Kürzel et al., 2022; Paulus et al., 2022) and fish (Mazzeo et al., 2008; Rossel et al., 2020). Recently, unsupervised methods for rapid delimitation in biodiversity assessments based on MALDI-TOF MS data were devised (Rossel and Martínez Arbizu, 2020; Renz et al., 2021) and applied in investigations of deep-sea biodiversity (Rossel et al., 20232b). Further studies have shown that the resolution of proteomic fingerprinting can go beyond mere species identification such das differentiation of some species on sex level (Rossel and Martínez Arbizu, 2019), developmental stage (Rossel et al., 2022a) and host species of important pathogen-vector species (Niare et al., 2016, 2018).
This makes MALDI-TOF MS a promising tool for species identification in Cnidaria but it has only been applied on selected cnidarian taxa so far including benthic stalked jellyfish (Staurozoa) (Holst et al., 2019), siphonophores (colonial pelagic Hydrozoa) (Park et al., 2021), and cold water corals (Anthozoa) (Korfhage et al., 2022). However, MALDI-TOF MS has never been tested for species identification by using the gelatinous tissues of scyphozoan and hydrozoan medusae before. In addition to these previous studies applying MALDI-TOF MS on selected cnidarian taxa, the present study aims to investigate mass spectra variability across various taxa using the so far largest data set of cnidarians including four classes (Scyphozoa, Hydrozoa, Staurozoa and Anthozoa). Beyond species classification, we furthermore investigate the influence of factors such as developmental stages and spatial origin on classification success at species level to evaluate the applicability of the method in future classification approaches.
Material and Methods
Samples
In total, 278 specimens of Cnidaria belonging to 23 different species from four classes were analyzed (Table 1). For four species (Aurelia aurita Linnaeus, 1758, Chrysaora hysoscella(Linnaeus, 1766), Cyanea capillata (Linnaeus, 1758) and C. lamarckii Péron & Lesueur, 1810) specimens of different ontogenetic stages (polyp, ephyra and medusa) were included (Table 1). Specimens were either sampled in the field or obtained from cultures (see supplementary table 1). Raw data of measurements from staurozoan species were obtained from (Holst et al., 2019). Field specimens were morphologically identified to species level by taxonomic experts immediately after collection, before complete specimens or subsamples were preserved in undenatured ethanol (80 - 96%). Cultures of scyphozoan polyps were reared from planulae which were obtained from sampled mature medusae; ephyrae from laboratory cultures were produced by strobilating polyps (supplement table 2).
Table 1: Number, developmental stage, and sampling region of analyzed specimens of species from four cnidarian classes. NS = North Sea, BS = Baltic Sea.