We sampled the three pipefish species, Nerophis ophidion, Syngnathus rostellatus and Syngnathus typhle in April and May 2016 by snorkelling in seagrass meadows around the Kiel Fjord (54°44'N; 9°53'E). All animals were kept for acclimatisation to laboratory conditions for two weeks in the aquaria at GEOMAR. Males were randomly assigned to one of the three reproductive stage: non-pregnant males, pregnant males and males after parturition. Only males assigned to be sampled at pregnant and after parturition stage were permitted to mate with the females. At mid pregnancy, males were either left in the tanks to be sampled after parturition or sampled immediately. Fish were killed by an overdose of MS222 and their weight and length were measured. Gills were collected and stored in RNAlater (Qiagen) for candidate gene expression analysis.RNA was extracted from gill tissue with RNeasy 96 Universal Tissue Kit (Qiagen) following the manufacturers protocol for animal tissues. RNA yield was measured by spectrometry (NanoDrop ND-1000; peQLab) and 200 ng/µl were used for reverse transcription with QuantiTect®Reverse-Transcription Kit (Qiagen). We used primer pairs for immune system related and metabolism related genes (Beemelmanns and Roth 2016 doi:10.1002/ece3.2391). The expression patterns of genes were measured using a Fluidigm-BioMarkTM system based on 96.96 dynamic arrays (GE-Chip).

Negative delta threshold cycle, quantitative polymerase chain reaction (-dCt values)