Chromosome Science 15: 23-25, 2012 Takeuchi et al. 23 Regular Article Chromosomes of two species of Japanese fishflies in the genus Parachauliodes (Megaloptera: Corydalidae: Chauliodinae) Yoshinori Takeuchi, Koji Iizuka, Hiroyuki Koishi, Takuzo Yamada and Hidehiro Hoshiba Received: August 18, 2011 / Accepted: May 17, 2012 © 2012 by the Society of Chromosome Research Abstract Introduction We analyzed chromosomes of two species of Japanese Insects known as dobsonflies or fishflies (Megaloptera: fishflies (Megaloptera: Corydalidae: Chauliodinae), Para- Corydalidae) are widely distributed around the world. In chauliodes continentalis and P. japonicus. The chromo- Japan there are 12 species belonging to three genera of some numbers in both species were 2n=20 consisting of Corydalidae (Liu et al. 2006; 2007a, b, 2008). 9 pairs of autosomes plus XX chromosomes in females The first chromosomal study of Asian Corydalidae was and Xy in males. The X chromosomes were subtelocen- made by Itoh (Itoh 1933a, b). Using a paraffin sectioning tric while the y was the smallest chromosome of the set. technique, he reported the chromosome number of the The sex chromosomes of first meiotic metaphase (MI) fishfly Chauliodes japonicus (=Parachauliodes japonicus, spermatocytes in both species invariably formed biva- at present) to be 2n=20 (i.e., 18 autosomes plus an XY lents synchronously with the autosomes and formed sex chromosome pair) and the dobsonfly Protohermes parachute-type bivalents, suggesting that the species in grandis to be 2n=23. Takeuchi et al. (2002) confirmed this genus share a common sex bivalent mechanism. the chromosome number of P. grandis to be 2n=24 using Key words: Chromosomes, fishflies, Parachauliodes, sex the air-drying technique improved by Kezer and Sessions chromosomes, Xy/XX, Xyp (1979). The diploid chromosomes of this species consist of 11 autosomes plus XY chromosomes in males and XX chromosomes in females. Hughes-Shrader (1980) observed the chromosome number of North American species, Neohermes fillicornis to be 2n=22 (i.e., 20+XY). In the present study karyotypes of two species of fishflies, Parachauliodes continentalis and P. japonicus, were analyzed using an air-drying technique improved by Hoshiba et al. (1989). Materials and Methods Thirteen larvae of Parachauliodes continentalis van der Weele 1909 were collected from the Sugihara River (35°05’N, 134°53’E) of Hyogo Prefecture, Honshu, Japan Yoshinori Takeuchi (*) in March to April of 1995, where this species is reported Bohkai Junior High School, Nishiakashi-minamimachi 1-1-33, to have a life cycle with larval stage extending from 2 to Akashi, Hyogo 675-0041, Japan 3 years (Takeuchi and Yamada 1999). Seven larvae of E-mail: [email protected] Parachauliodes japonicus (McLachlan 1867) were collected Koji Iizuka from the Sugihara River (35°11’N, 134°55’E), the Kako Takashima 2nd Junior High School, Takashimadaira 2-24-1, River (35°13’N, 134°57’E) and the Kizu River (34°44’N, Itabashi-ku, Tokyo 175-0082, Japan 135°06’E), all in Hyogo Prefecture, Honshu, in March to Hiroyuki Koishi April of 1995. Hosei University Daini Junior High School, Kidukiohmachi 6-1, These larvae of two species appeared to be near their final Kawasaki, Kanagawa 211-0031, Japan instar of development. Chromosome slides were prepared Takuzo Yamada by means of an air drying method (Hoshiba et al. 1989). In Nagoya University of Art, Kumanosho Furui 281, Kitanagoya, P. continentalis, 10 spermatogonial metaphase cells from 4 Aichi 481-8502, Japan males, 10 oogonial cells from 5 females, and 6 first meiotic Hidehiro Hoshiba metaphase (MI) cells from 3 males were examined. In P. Graduate School of agriculture, Tamagawa University, Machida, japonicus 10 spermatogonial metaphase cells from 5 males, Tokyo 194-8610, Japan 10 oogonial metaphase cells from 2 females and 2 MI cells 24 Chromosomes of two species in the genus Parachauliodes Table 1. Chromosome numbers of species of Corydalidae (Megaloptera) so far studies with karyotype descriptions. Species Chromosome morphology Method Authors number (2n) Autosomes X Y CORYDALINAE (Dobsonflies) Protohermes grandis 23 1LSM+2M+7T α-? β1, β2‐dot paraffin Itoh 1933b Protohermes grandis 24 1LSM+2M+7T+1dot SM dot drying-1*2 Takeuchi et al. 2002 Corydalus cornutus 24 1LM+1M+8T+1dot SM dot squash Hughes-Schrader 1980 CHAULIODINAE (Fishflies) Neohermes fillicornis 22 10M M dot squash Hughes-Schrader 1980 Parachauliodes continentalis 20 1LSM+1M+7T ST dot drying-2*3 Present study Parachauliodes japonicus*1 20 1LSM+1M+7T SM dot paraffin Itoh 1933a Parachauliodes japonicus 20 2SM+4M+3ST ST dot drying-2*3 Present study *1 = reported as “Chauliodes japonicus” in Itoh (1933a). However, this species might be misidentidication of P. continentalis (see text). *2 = method by Kezer and Sessions (1979) *3 = method by Hoshiba et al. (1989) metaphase from 2 males were also examined. In addition to spermatogonial metaphase plates, several cells in meiotic divisions were also available from testes of male larvae as shown here, although Itoh (1933a) stated that no meiotic divisions are observed in larval stages. The karyotypes were described following the nomenclature of Levan et al. (1964). Results and Discussion The chromosome number P. continentalis was 2n=20 (9 autosomal pairs +XX in the female and 9 autosomal pairs +Xy in the male, Fig. 1A). The autosomes consisted of one pair of extremely large submetacentric chromosomes, one pair of moderately large metacentric chromosomes, and 7 pairs of telocentric chromosomes. The sex chromosomes consisted of subtelocentric X and the smallest telocentric y (Table 1). At diakinesis, 9 autosomal bivalents and one bivalent with a ‘‘parachute’’ shape traditionally designated by the symbol Xyp were found (Fig. 2A). This sex bivalent was initially termed ‘parachute-like’ association by Stevens (1906) among the coleopteran insects as the X and small Y chromosomes form a canopy like sexual bivalent. Smith (1950) symbolized this as Xyp. Figure 1. Male and female karyotypes from spermatogonial and The chromosome number P. japonicus was 2n=20 (9 oogonial metaphases of two fishfly species of the genus Para- autosomal pairs +XX in the female and 9 autosomal pairs + chauliodes (male, upper; female, lower). A: Parachauliodes conti- Xy in the male, Fig. 1B). The autosomes consisted of 2 pairs nentalis (2n=20). B: P. japonicus (2n=20). Bar represents 5μm. of submetacentrics, 4 pairs of metacentrics, and 3 pairs of subtelocentric chromosomes. The sex chromosomes consisted of a subtelocentric X and the smallest telocentric that were collected from Miyagi and Saitama Prefectures, y (Table 1). At diakinesis, 9 autosomal bivalents and an Xyp Honshu, and identified by Dr. Hanjiro Okamoto, leading bivalent were found (Fig. 2B). No significant differences taxonomist of the group in Japan at that time, were actually were found among karyotypes of three populations from not “C. japonicas (now Parachauliodes japonicus)” but three different rivers in Hyogo Prefecture. P. continentalis. This could have easily been done since Itoh (1933a) reported that the karyotype of Chauliodes the larval morphology of P. japonicus and P. continentalis japonicus (=Parachauliodes japonicus) obtained from extremely resemble each other, and as mentioned above, larvae consists of one pair of extremely large chromosomes these two species can be obtained in the same rivers in with ‘‘sub-terminal attachment’’ (i.e., subtelocentric Miyagi and Saitama Prefectures (even though they are not centromeres), one pair with ‘‘median attachment’’ strictly sympatric). (metacentric), 7 pairs with ‘‘terminal attachment’’ In the North American fishfly Neohermes fillicornis (telocentrics), and one pair of heteromorphic sex (2n=22), all autosomes were metacentric (Table 1; Hughes- chromosomes: the X chromosome was rod-shaped with a Shrader 1980), and the chromosome numbers of the other constriction at a ‘‘sub-terminal’’ (i.e., subtelocentric) point, four North American species of fishflies were either 2n=20 and the Y chromosome was tiny round blob (Table 1). or 22 (Takeuchi, unpublished data). In contrast, most of the The karyotype of “C. japonicus” reported by Itoh (1933a) autosomes of P. continentalis were telocentric. appears to be identical to that of P. continentalis reported The sex chromosomes of Neohermes fillicornis were here. Thus, it is likely that materials used by Itoh (1933a) the smallest of the set with the Y being minute (Hughes- Takeuchi et al. 25 Figure 2. Male MI spermatocyte of Parachauliodes con- tinentalis (A) and P. japonicus (B). Arrows indicate X and y chromosomes that form parachute-type bivalents (Xyp). Bar represents 5μm. Shrader 1980). On the other hand, the two Japanese talis species-group (Megaloptera: Corydalidae). Zootaxa 1439:1-46 species of fishflies in the genus Parachauliodes (2n=20) Liu XY, Hayashi F, Yang D (2007b) Revision of the Neochauliodes si- had X chromosomes that were not minute but were larger nensis species-group (Megaloptera: Corydalidae: Chauliodinae). than the 9th autosomal chromosome. It is possible that Zootaxa 1511:29-54 Liu XY, Hayashi F, Yang D (2008) Systematics and biogeography of the this difference is from either a fission or fusion of the X fishfly genus Parachauliodes (Megaloptera: Corydalidae) endemic chromosome. to the East Asian islands. System Entomol 33:560-578 In MI spermatocytes of the two Japanese Parachauliodes Smith SG (1950) The cyto-taxonomy of Coleoptera. Can Entomol fishflies, the sex chromosomes invariably formed a bivalent