Chromosome preparation and karyotype analysis

Sampling

The mature sperms and oocytes were obtained by dissecting the nephridia of the healthy U.unicinctus, respectively. Artificial fertilization was conducted by mixing sperms and oocytes at a ratio of 10:1 in filter seawater (FSW), and then these fertilized eggs were cultivated until hatched in FSW (19.7 ± 0.3 °C, salinity 29 PSU, pH 8.29 ± 0.02). The hatched trochophores were collected using a 500 mesh sieve.

Chromosome preparation and karyotype analysis

Chromosomes of U.unicinctus trochophores were prepared as described by Earley (1975) with some modifications. The larvae were incubated in FSW containing 0.02% colchicine for 2.5 h at room temperature, and then transferred into Ca²⁺/Mg²⁺-free Dulbecco’s phosphate buffered saline (CMF-DPBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄×7H₂O, 1.1 mM KH₂PO₄) for 30 min with continuous mild shaking to obtain the dissociated larval cells. Successively, the cells were treated with 0.075 mol/L KCl for 30 min, collected by centrifuging at 1200 g for 5 min, and then fixed three times with cold Carnoy’s fixative (3 ethanol: 1 glacial acetic acid) for 15 min each. After centrifugation, the cells were re-suspended in 50% glacial acetic acid aqueous solution, and then dropped onto preheated clean glass slides at 56 °C, and air-dried. Finally, these samples were stained with 10 µg/ml 4’, 6-diamidino-2-phenylindole (DAPI) (Solarbio, China) for 15 min, and were examined and photographed under a Nikon Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).

The chromosome lengths, chromosome relative lengths and arm ratios from well-formed chromosomes in metaphase were measured and calculated using MICRO-MEASURE 3.3 software. Data were presented as mean ± SEM (n = 5). Chromosomes were classified according to the description of Levan et al. (1964), and the homologous chromosome was assigned based on the similarities in length and centromere position using PHOTOSHOP CS6 software. The idiogram was constructed according to the arm ratio and relative length of the chromosomes. The karyotype was classified as described by Stebbins (1971).