Contributions to Zoology, 80 (1) – 2011Pawel Koperski; Rafal Milanowski; Agnieszka Krzyk: Searching for cryptic species in Erpobdella octoculata (L.) (Hirudinea: Clitellata): discordance between the results of genetic analysis and cross-breeding experiments
Material and methods

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Experimental mating

Morphologically divergent individuals were bred in the laboratory during long-term experimental mating. 84 parent pairs (168 individuals) of dark, medium and very light forms, collected in lakes Łaśmiady and Wadąg and in the rivers Wisła (Vistula) and Święcek were allowed to reproduce in different combinations for 7 months (June-December). Each pair was separated in polypropylene, cylindrical containers, 0.25 dm3 in volume. A fragment of the wall in each container (20 cm2) was replaced with a plastic net with 1mm mesh-size. Each 10 containers were submerged in aquaria filled with circulating, filtrated and aerated water at the temperature of 20°C and with constant photoperiod of 16 hrs of light and 8 hrs of darkness. The animals were fed once in two days with live larvae of Chironomidae with regular addition of frozen Tubifex, Gammarus and fish meat.

The numbers of cocoons laid and number of hatchlings of each pair were counted. Young individuals were moved to another set of polypropylene containers with the volume of 0.25 dm3 with filtered water at the temperature of 20°C and with constant photoperiod of 16 hrs of light and 8 hrs of darkness. Young leeches were fed daily with live Enchytraeidae and small Chironomidae. The quantity of offspring of each pair which remained alive after 5 days was recorded. The morphological form of each young individual was determined as soon as it was (15-20 days after hatching). The significance of differences between values of demographic parameters were tested with Wilcoxon’s test. The number of cocoons with dead or no embryos was high, as well as mortality, which was high, mainly because of cannibalism.