Embryo Developmental Stages of E. kanangrensis

Female specimens of E. kanangrensis were collected in Kanangra-Boyd National Park, New South Wales, Australia. To obtain all developmental stages, we dissected developing embryos of various stages in the months from September to December. Each female carries up to 100 embryos, representing a series of developing stages (sometimes even ranging from the one-cell stage up to the fully developed embryo). The chorion and vitelline membrane were removed by hand with Dumont size 5 forceps and directly afterwards the embryos were fixed in 4% formaldehyde in 0.1 M phosphate-buffered saline with 0.1% Tween-20 (PBST) (pH 7.4) for four to six hours at room temperature. Embryos were then dehydrated stepwise into 100% methanol and stored at −20°C for at least three weeks before being used for in-situ hybridization experiments.