Materials and Methods
Snails and slugs: materialsnext section
Stylommatophoran sperm cells are most easily obtained from the spermatophores that these hermaphroditic animals produce and transfer during mating. Therefore, adult, large-bodied snails and slugs were collected in several locations in the Dutch provinces of Zuid-Holland, Noord-Holland, and Flevoland during spring and early summer (when mating takes place) of 2014 and 2015. We collected animals that were found in copula, but also mature animals that may have recently mated and therefore contain fresh sperm in the spermatophore-receiving organ. All specimens were stored in 70% ethanol in the field. The animals were identified, dissected, and, when spermatophores were encountered, these were carefully removed and cut or broken lengthwise. The exposed halves were then dehydrated in an automated Leica EM CPD300 critical-point drier and affixed on stubs for scanning electron microscopy.
We used Passeriformes sperm samples that had been preserved in 5% formalin at the Department of Animal and Plant Sciences, University of Sheffield. These samples had been obtained for previous studies, using a range of techniques (as detailed in Birkhead et al., 1993; Immler and Birkhead, 2005, 2007). First, samples were cooled at 5°C overnight. Then, 10 µl of the pellet of the sample was placed directly on SEM stubs and air-dried for scanning electron microscopy.
Scanning electron microscopy
Sperm samples were coated with platinum/palladium (2 nm and 20 nm in the case of bird and snail sperm cells, respectively). Then, they were viewed in a JEOL JSM-7600F FEG-SEM, at magnifications up to 20,000×. For each sample, we attempted to view and photograph at least 20 sperm cell heads. Coiling direction was determined by eye for each sperm cell. Using the letters embossed on the stub holders, it was confirmed that the image is not mirrored at any stage during the imaging process.