Stability of proteomic signals
To specify a potential stable species gap in proteomic composition, we compared the intra- and inter-specific variability of proteomic fingerprints using Euclidean distance as measure. A distinct gap at a Euclidean distance of approx. 0.8 occurred when intra-specific variability was minimized by excluding variation between samples, seasons, and regions. A similar threshold between inter- and intra-specific distances was observed e.g. for calanoid deep-sea copepods (Renz et al., 2021). Intra-specific variability increased, when specimens from different regions or sampling seasons were included leading to a stronger overlap of the maximal intra-specific and minimal inter-specific distance. While also sample history, e.g. sample storage conditions (temperature, pH, organic material in sample etc.) may impact proteomic spectra (Rossel and Martinez Arbizu 2018b), the narrowing of the species gap is most likely mainly driven by changes in proteomic spectra based on population specific patterns and environment-induced variations in cell composition. In line with this interpretation of our data, proteomic patterns of mosquitos discriminated between colonies (Müller et al., 2013) and those of ticks varied with season and habitat (Karger et al., 2019). Strongest intra-specific distances, on the level of species distance, were observed between specimens from the brackish Baltic Sea and those from other regions in i.e. A. longiremis , C. hamatus and T. longicornis. In these cases, species identification by means of a universal classification approach seems to come to its limits. A larger number of Baltic Sea specimens could not be determined unambiguously based on a database only containing North Atlantic animals. Apparently, salinity has a quite strong additive effect on proteomic patterns compared to other factors. This seems conclusive as copepods have been found to change protein expression not only under thermal stress (Rahlffs et al., 2017) and over the seasonal cycle (Semmouri et al., 2020) but also under osmotic stressful conditions (DeBiasse et al., 2018). Copepods are capable to osmoregulate and change the osmolarity of the hemolymph (Roddie et al., 1984, Lee et al., 2012). Specifically marine copepods in brackish environments need to permanently control osmotic and cellular volume (Dutz & Christensen, 2018). Although most of the so far described changes in functional proteins are in the size fraction larger than measured by MALDI-TOF MS, it seems realistic that changes in cell physiology will become visible to some extent in the proteomic fingerprint from 2-20 kDa.
In addition to a physiological response, the observed pattern could also be due to a population-specific genetic aspect. The Baltic Sea was suggested to act as diversification hotspot to many native inhabitants (Geburzi et al., 2022) and a reduced gene flow between North and Baltic Sea populations was observed (Sjöqvist et al., 2015). However, to our knowledge, no information is available on population genetics and connectivity of Baltic A. longiremis , T. longicornis ,C. hamatus with the North Sea, the North Atlantic and Arctic populations. Further field and experimental studies will be necessary and useful to disentangle the multiple effects of environment, ontogeny, and underlying genetic variation on proteome spectra and to assess their impact on the ability to discriminate at the species level.