An immunolabelling technique to track sperm from different mates in the female reproductive organs of terrestrial gastropods

The mechanisms of sperm transfer, storage, utilization and digestion are crucial for understanding processes of postcopulatory sexual selection. Previous studies analysing postcopulatory processes have generally focused only on the ultimate outcome of the interactions between male and female sexual selection (paternity patterns). For a mechanistic understanding of the fate of received sperm and the involved patterns of postcopulatory sexual selection new techniques are required. Here we present an improved immunolabelling technique to track the fate of 5-bromo-2'-deoxyuridine (BrdU)-labelled sperm in the female reproductive organs of gastropods. The technique was tested in individuals of the simultaneously hermaphroditic land snail Arianta arbustorum (Linnaeus, 1758). We determined the percentage of labelled sperm in spermatophores delivered and assessed the reliability of detecting labelled sperm in the spermatheca (sperm storage organ) and bursa copulatrix (sperm digestion organ) of the recipients. In our tests, the proportion of sperm labelled among the sperm produced by an individual averaged 99.3%. Furthermore, labelled sperm could be consistently visualized in both the sperm storage and sperm digestion organ of all recipients examined. Combined with a traditional sperm staining technique, we showed that the BrdU-labelling technique allows us to distinguish between sperm from two males in the female reproductive tract of double-mated snails. In a controlled growth experiment, we found that repeated BrdU-application slightly retarded shell growth of juvenile snails, but did not influence their adult size, survival, number of sperm delivered and mating frequency. The technique presented could be adjusted to other gastropod species providing insight into mechanisms of sperm competition and cryptic female choice.