Introduction
Marine zooplankton species are useful indicators of environmental
variation and climate change as they rapidly respond to changes in
biological and physical conditions. Awareness of the importance of
time-series based zooplankton monitoring is increasingly growing. Time-
and cost-efficient species identification methods are a strong need not
only for time-series but for many fields of marine science, e.g.
assessment of community turnover or biodiversity in the context of
ecosystem-based management. Manual counts of these small organisms
require well-trained personnel, as the taxonomic resolution is often
very limited due to high morphological similarity or absence of
diagnostic features in young developmental stages. Although taxonomic
expertise remains a keystone for community monitoring, integration with
molecular approaches can enhance and accelerate identification
processes. However, DNA barcoding of single organisms is to date not
suitable for routine species identification in time-series as it
requires numerous steps in the working procedure accompanied with high
costs. Genetic multi-species approaches such as organismal metabarcoding
of bulk samples are finding their way more and more into zooplankton
monitoring (Bucklin et al., 2016, 2019, 2021 and references therein) as
they combine comprehensive information on species occurrences with a
good methodological efficiency (Laakmann et al., 2020). Also approaches
focusing on environmental DNA metabarcoding are getting increasingly
applied (Djurhuus et al., 2020, Suter et al., 2020). Although these
multispecies approaches provide valuable extensive species information,
they remain semi-quantitative so far.
Proteomic fingerprinting as a fast, efficient, and low-cost method for
species identification (Rossel et al., 2019, Renz et al., 2021) has a
large potential to evolve to a valuable add-on to the current classical
and molecular toolbox in zooplankton identification. In short, sample
tissue is extracted in a matrix-solution, which is then applied onto a
target plate. The extracted compounds, mainly consisting of small
cytosolic proteins and peptides (Ryzhov & Fenselau, 2001) are measured
by matrix-assisted laser desorption ionization time of flight mass
spectrometry (MALDI-TOF MS) producing species-specific mass spectra,
allowing the differentiation and, in combination with a reference
database, the identification of specimens. Within the last ten years
several proof-of-concept studies on metazoans revealed its general
applicability for invertebrates (Murugaiyan & Roesler, 2017),
specifically for insects (El Hamzaoui et al., 2018, Chavy et al., 2019,
Lawrance et al., 2019, Hasnaoui et al., 2022) and arachnids (Diarra et
al., 2017, Karger et al., 2019, Gittens et al., 2020, Ngoy et al.,
2021), which are relevant as vectors and pests. Far less studies address
taxa less relevant to human health. In the aquatic realm, research is
mainly focused on groups which hold pivotal positions in marine and
limnic food webs, i.e. copepods (Riccardi et al., 2012, Laakmann et al.,
2013, Bode et al., 2017, Kaiser et al., 2018, Rossel & Martinez Arbizu,
2019, Yeom et al., 2021, Renz et al., 2021), cladocerans (Hynek et al.,
2018) and fish (Volta et al., 2012, Maasz et al., 2017, Rossel et al.,
2021).
To advance the method on its way to becoming a standard tool for
zooplankton identification, a thorough evaluation of the sensitivity and
specificity of proteomic fingerprinting and the intra-specific variance
of fingerprints is essential. For bacteria it was shown that culture
conditions may influence peak numbers, spectrum quality and
identification success based on MALDI-TOF MS (Goldstein et al., 2013,
Balazova et al., 2021). Knowledge on spectra variations and factors
influencing them is limited in metazoans, e.g. spectra of ticks varied
with season and habitat (Karger et al., 2019) and population-specific
patterns were identified for bed bugs (Benkacimia et al., 2020) and
mosquitoes (Müller et al., 2013). However, underlying causes of
differences between regions, being either of genetic or environmental
origin, remain unclear. To our best knowledge, there is to date no
information on seasonal and regional variability of proteomic patterns
in marine invertebrates, their resilience against physiological or
environmental impacts or the stability of markers between genetically
more distant populations. As a first approach to these questions, we
analyzed mass spectra of abundant epipelagic copepods from different
zooplankton monitoring sites around the Atlantic and adjacent seas
covering a wide spectrum of environments from Arctic to temperate zones,
brackish and euryhaline waters as well as neritic and oceanic regimes.
The aim of our study was, based on data from various marine copepod
populations and species (i) to validate the general robustness of the
species differentiation and identification approach to data processing
and data variance, (ii) to determine specificity and sensitivity of
single proteomic markers for the species (iii) to estimate the
discriminatory power of proteomic fingerprinting and its sensitivity to
phylogenetic distance, (iv) to estimate inter- and intra-specific
variability of spectra searching for stable species gaps and the impact
of variation on identification success and finally (v) to present
perspectives of proteomic fingerprinting for marine zooplankton studies.