C 2000 The Japan Mendel Society Cytologia 65:351-358, 2000 C-Banded Karyotypes of Some Podisminae Grasshoppers (Orthoptera, Acrididae) from Japan Alexander G. Bugrov1,*, Elzbieta Warchalowska-Sliwa2, Gen lto3 and Sin-ichi Akimoto3 1 Novosibirsk State University, 630090 Novosibirsk and Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences, 630091 Novosibirsk, Russia 2 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, 31-016 Krakow, Poland 3 Hokkaido University, Faculty of Agriculture, Sapporo, 060-8589 Japan Accepted February 24, 2000 Summary C-banding patterns in Japanese grasshoppers belonging to the subfamily Podisminae: Podisma sapporensis Shiraki (2n •‰=23, NF=23), Parapodisma subastris Huang, Parapodisma ten- ryuensis Kobayashi, Parapodisma yamato Tominaga et Storozhenko, Parapodisma mikado (I. Boli- var), Fruhstorferiola okinawaensis (Shiraki) (2n d=21, NF =21), and Sinopodisma punctata Mist- shenko (2nd = 21, all chromosomes are two-armed) were studied. Cytogenetic similarities and differ- ences between particular species are discussed using the C-banding method. Key words Podisminae grasshoppers, Chromosome, Karyotype, C-banding. Karyotypes of 8 species of grasshopper belonging to the subfamily Podisminae have been de- scribed from Japan (Inoue 1985). However, the C-banding technique was not commonly used in the cytogenetic studies of Japanese Podisminae. Nevertheless, it has been used extensively with Or- thoptera to discriminate individual chromosomes and their behaviour during meiosis (Gosalvez et al. 1997). The application of this technique has produced new cytogenetic markers for comparative karyology of Podisminae (Westerman and Hewitt 1985, Bugrov et al. 1994, Bugrov 1995, Bugrov and Sergeev 1997). Moreover, the C-banding method gives information about the rates and features of chromosome evolution in groups of closely related species (Cabrero and Camacho 1986). It is important especially for the groups with geographically limited areas of distribution as, for exam- ple, the genus Parapodisma Mistshenko. The aim of the present work was to study unknown karyotypes and C-banding features of some Podisminae species from Japan. Material and methods Seven species of Podisminae belonging to the fauna of Japan were studied (Table 1) . Male insects were injected with 0.1% colchicine for 1.5-2 h. The testes were fixed in glacial acetic alcohol (1 : 3), and kept in 70% ethanol. Air dried preparations were made by squashing the testicles in 45% acetic acid and freezing them in dry ice. They were then stained with C-banding by treating with 0.2 N HCl for 15-20 min, immersed in a saturated solution of Ba(OH)2 at 61•Ž for 3-5 min, rinsed in water, immersed in 2•~ SSC at 61•Ž for 60 min , rinsed, air-dried, and stained with 2% Giemsa. Each portion of the eggs was stored in a separate Petri dish with moist sand and kept in an in- cubator at room temperature. After 15-20 days of incubation, the eggs were placed in a solution of 0.05% colchicine in insect saline and the tops of the nonmicropylar end were removed . They were then incubated at 30•Ž for 1.5-2 h. The embryos were then dissected out of the eggs into 0.9% * Corresponding author . 352 A. G. Bugrov et al. Cytologia 65 Table 1. List of taxa and collection localities sodium citrate solution for 20-30 min at room temperature prior to fixation in glacial acetic alcohol (1 : 3) and air-dried squashed preparations were made. They were stained using the C-method simi- lar to that for testicles, with minor modifications. Results Podisma sapporensis Shiraki. The chromosome complement of P. sapporensis from the popu- lation under study consists of 2n d= 23. All autosomes are acrocentric and can be divided into 3 size groups: 2 long (L1, L2), 6 medium (M3—M8), and 3 short (S9—S11). The size of the acrocentric X chromosome is approximately similar to the M4 pair. The paracentromeric C-blocks occur in all chromosomes of the complement, their size varying among autosome pairs. The M4 and M9 pairs of autosomes were characterised by the presence of distal C-bands. Additionally, the S9 pair of auto- somes has an interstitial C-band between the paracentromeric and telomeric C-blocks (Fig. la). In a few males the X chromosome has in addition to the centromeric C-band a minute interstitial C-band near the paracentromeric one (Fig. lb). In the prophase of meiosis the long autosomes form 2-4 chiasmata, medium-1-2, and the short autosome only one chiasma. The mean frequency of chias- mata on a cell is .X=17.64 (-±1.18). Parapodisma subastris Huang. 2n •‰=21. All chromosomes are acrocentric. They may be di- vided into three size groups; 2 long (L1, L2), 7 medium (M3—M9) and 1 short (S10). The size of the acrocentric X chromosome is approximately similar to the M4 pair. The paracentromeric C-blocks occur in all the chromosomes of the complement. The chromosomes of the M4 pair have a minute telomeric C-block. The autosomes of the M6 pair have a thick telomeric C-block and interstitial C- band in heteromorphic state near the telomeric one (Fig. 2). In the prophase of meiosis the long au- tosomes form 2-3 chiasmata, medium-1-2, but the short bivalent only one. The mean frequency of chiasmata on a cell is 12.90 (•}1.00). Parapodisma yamato Tominaga of Storozhenko. 2n d=21. All chromosomes are acrocentric. They may be divided into three size groups: 2 long (L1, L2), 7 medium (M3—M9), and 1 short (S10). The size of the acrocentric X chromosome is approximately similar to that of the M4 pair. The para- centrometric C-blocks occur in all the chromosomes of the complement. The chromosomes of the M5 pair revealed telomeric C-blocks. In the prophase of meiosis the large telomeric C-blocks in the M6 pair of autosomes seem to become double (Fig. 3) similarly to P tenryuensis (see below). In the prophase of meiosis the long autosomes form 2-3 chiasmata, medium-1-2, and short bivalent only one. The mean frequency of chiasmata on a cell is X=13.06 (•} 1.08). Parapodisma tenryuensis Kobayashi. 2n •‰= 21. In 4 males all chromosomes are acrocentric. They amy be divided into 3 size groups: 2 long (L1, L2), 7 medium (M3—M9), and 1 short (S10). The size of the acrocentric X chromosome is approximately similar to that of the M4 pair. The paracen- tromeric C-blocks occur in all the chromosomes of the complement. The L1 pair of chromosomes 2000 C-banded karyotypes of Podisminae grasshoppers 353 Figs. 1-4. I a, b. Podisma sapporensis. Diakinesis. a) M4 with distal C-band (thin arrow) additionally, S9 with interstitial C-band (thick arrow). b) X chromosome with minute intercalar C-band near paracen- tromeric one (thick arrow). 2. Parapodisma subastris. Diakinesis. M4 with minute telomeric C-block (thin arrow), M6 with thick telomeric C-block (thin arrow) and interstitial C-band in heteromorphic state near the telomeric one (thick arrow). 3. Parapodisma yamato. Diakinesis. M5 and M6 with telomeric C- bloks (thin arrows). 4a-d. Parapodisma tentyuenis. a) Diakinesis. L1 with minute telomeric C-block and M6 with thick C-block (arrows). b) Metaphase II. M7 acrocentric with minute interstitial C-band (thick arrow). c) Heteromorphic M7 bivalent (thin arrow). d) Metaphase II with acrocentric (on the right) and metacentric (on the left) M7 bivalent (thin arrows). Bar equals 10 um. 354 A. G. Bugrov et al. Cytologia 65 has a minute telomeric C-block. In the prophase of meiosis the large telomeric C-blocks in the M6 pair of autosomes seem to become double (Fig. 4a). In metaphase II a clearly visible euchromat- ic zone contrasts sharply with the telomeric and near telomeric interstitial C-block in this chro- mosome. The M, pair is acrocentric with a large centromeric C-block and minute interstitial one (Fig. 4b). In the prophase of meiosis the long au- tosomes form 2-3 chiasmata, medium-1-2, and short bivalent only one. The mean frequency of chiasmata on a cell is x=13.78 (•}1.06). One male demonstrated chromosome het- eromorphism in the M7 pair. At prophase I the M7 bivalent consists of one acrocentric homo- logue with the large centromeric C-block and a minute interstitial one. The other homologue in this bivalent is of the same size, metacentric with very small centromeric and telomeric C-blocks in one of the arms. This heteromorphic bivalent forms only one terminal or subterminal chiasma (Fig. 4c). At first anaphase the M7 bivalent un- dergoes divisions as in other autosomes. At sec- ond metaphase the cells with the acrocentric M7 and cells with the metacentric M7 were clearly seen (Fig. 4d). Parapodisma mikado (I. Bolivar). 2n d = 21. The karyological analysis was performed on neuroblast mitosis of embryos. The chromosome complement 2n =21, with all acrocentrics, possi- bly shows the male karyotype (2n d=21, Fig. 5a). They may be divided into 3 size groups: 2 long (L1, L2), 7 medium (M3—M9), and 1 short (S10). The size of the acrocentric X chromosome is approximately similar to the M5 pair. The paracentromeric C-blocks occur in all the chro- mosomes of the complement. The M5 chromo- Figs. 5-6. 5a-c. Parapodisma mikado. a) C-banded karyotype, b) mitotic metaphase. M5 pair with large inter- some pair has a small paracentromeric C-block, stitial C-bands near paracentromeric and telomeric regions a large interstitial one near the telomeric region (thick arrow). c) Thin C-bands between paracentromeric and a telomeric C-block (Fig. 5a, b). In addition, and thick interstitial C-blocks (thick arrows).