SHORT COMMUNICATION Genome sizes of three species in the subtribe Carabina (Coleoptera: Carabidae) Teiji SOTA 1 , Junji KONUMA 1 , Mayuki FUJIWARA 2 and Eiichi SHOGUCHI 2 1 Department of Zoology, Graduate School of Science, Kyoto University, Kyoto, Japan and 2 Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan Abstract Genome sizes of three species in the subtribe Carabina (Coleoptera: Carabidae: tribe Carabini) were estimated from nuclei of spermatozoa using a flow cytometry. The estimated haploid genome sizes were about 330 megabases (Mb) for Damaster blaptoides, about 280 Mb for Carabus uenoi and about 260 Mb for Carabus yamato. Key words: Carabus uenoi, Carabus yamato, Damaster blaptoides, flow cytometry, spermatozoa. Information of the genomic size of an individual organ- ism is useful in many aspects of genetic and genomic studies. The beetles of the subtribe Carabina (=Carabus sensu lato; Deuve 2004) are an intriguing group for various evolutionary studies (e.g., Sota & Nagata 2008; Sasabe et al. 2010; Konuma et al. 2011), and the knowl- edge of their genomic sizes is essential for analyzing genetic basis of diverse traits. Here we estimated genome sizes of three species in the subtribe Carabina, which are particularly interesting for evolutionary studies. Mature spermatozoa were collected from one male beetle each of Damaster blaptoides fortunei (Adams) from Awashima I., Niigata Pref., Japan (collected in July 2011 by J. Konuma), Carabus (Ohomopterus) uenoi (Ishikawa) and C. (O.) yamato (Nakane) from Mt. Kon- gosan, Osaka, Japan (collected in June 2011 by T. Sota). The spermatozoa were used to estimate genome size. Mature testes were removed from males and preserved in a freezer at -28°C until sample preparation. After melting, a testis was torn into pieces and crushed in 200–300 mL physiological saline with RNase using forceps. The whole suspension was transferred to a 1.5-mL tube and vortexed for 10 s to liberate sperma- tozoa from sperm bundles. Then the suspension was filtered through 30-mm-diameter mesh nylon filter to remove large particles other than spermatozoa. The genome size analysis was conducted using a BD FACSAria II cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). A 50-mL aliquot of the above suspen- sion was mixed with 100 mL phosphate buffered saline containing 0.1% Tween 20 (PBST) and stirred in an ultrasonicator for 10 s to dissociate attaching spermato- zoa. The suspension was filtered through 35-mm- diameter mesh nylon filter. A 50-mL suspension (2–4 ¥ 10 4 cells/mL) was mixed with 1 mL NIM-DAPI (#731085; Beckman Coulter, Brea, CA, USA) to stain nuclei of spermatozoa and left at room temperature for 5 min. This sample was filtered through 35-mm-diameter mesh nylon filter again and then analyzed by a BD FACSAria II cell sorter using detector setting Violet-A with the 407-nm violet laser to determine the fluorescent intensity of a spermatozoa nucleus. For calibration, spermatozoa of Takifugu rubripes (haploid genome size of 365 megabase pairs (Mb)) were used as the size stan- dard. Flow cytometry measurements were conducted for both individual samples with and without the internal standard (T. rubripes) mixed. Flow cytometric measurement of each sample resulted in peak fluorescent intensity of 3833 for D. blaptoides, 3315 for C. uenoi and 3087 for C. yamato (Fig. 1). Mea- surements for mixed samples gave consistent results: 3831 for D. blaptoides, 3203 for C. uenoi and 2977 for C. yamato (Fig. 1). Estimated genome sizes based on the comparison with control haploid nuclei of T. rubripes were 328 Mb for both measurements of D. blaptoides, Correspondence: Teiji Sota, Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan. Email: sota@terra.zool.kyoto-u.ac.jp Received 21 May 2012; accepted 9 July 2012. Entomological Science (2012) doi:10.1111/j.1479-8298.2012.00541.x © 2012 The Entomological Society of Japan