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.