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.