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.