No. 5] Proc. Japan Acad., 61, Ser. B (1985) 215 60. A Study o f the H®locentric Chromosomes by a Simple Method for Differential Stain By Naoto MURAMOTO Hakodate La Salle High School, Hakodate, Hokkaido 041 (Communicated by Saj iro MAKINO, M. J. A., May 13, 1985) The chromosomes have ordinarily a single centromere, however, holocentric (holokinetic or diffuse centromeric) chromosomes have been known in some ani- mals and plants (White 1973) . The holocentric chromosomes have been presented mainly throughout the hemipteran insects (Hemiptera) . They were first found by Hughes-Schrader and Ris (1941) in coccids (Hemiptera ; Homoptera) . Since then, the holocentric chromosomes have been found in insects of orders Anoplura, Dermaptera, Hemiptera, Lepidoptera, Mallophaga and Odonata (Bayreuther 1955 ; Hughes-Schrader 1945 ; Lewis and Scudder 1957 ; Maeki and Hayashi 1979; Muramoto 1974, 1976; Nur 1978; Ortiz 1969; Scholl 1955; Schrader 1947; Takenouchi and Muramoto 1971; Ueshima 1979; White 1973) . Such chromosomes may become attached to the spindle fibers throughout their entire length, and during the anaphase of the mitosis they ordinarily move to each pole keeping their long axis parallel to the metaphase plate. In addition to this, the fragment of the holocentric chromosome has been known to behave in a way similar to the normal chromosome during the cell division. In general, the holocentric nature has been elucidated by the chromosome behavior during the cell division as stated before. In Dysdermus intermedius (Heteroptera), Rithmann and Permantier (1973) gave a proof of that by the electron-microscopic observation in which a holocentromere consists of discrete centromeres. Recently various staining methods for the banding patterns of the chromo- somes have been devised by many investigators (see, Makino 1979; Nakagome 1982; Yosida 1979). Tanaka and Ohta (1982), and Utsumi (1977) were especial- ly successful in differential staining of the centromeric regions of the plant chromosomes. The present paper deals with the differential staining according to the original method and the holocentric chromosomes of insects. Materials. Male germ-cells of the following species were used. Colleoptera; Aulacophora f emoralis Motschulsky (Chrysomelidae). Dermaptera ; For ficula mikado Burr (Forficulidae). Hemiptera; Cicadella viridis Linne (Homoptera, Cicadellidae), Lygaeus equestris Linne (Heteroptera, Lygaeidae), Furydema rugosa Motschulsky (Heteroptera, Pentatomidae), Palomena angulosa Motschulsky (Heteroptera, Pentatomidae ), Plautia stali Scott (Heteroptera, Pentatomidae). Lepidoptera; Abraxas sp. (Geometridae). Orthoptera; Oxya yezoensis Shiraki (Locustidae), Podismamikado (Locu- stidae). Method. (1) Pre-treatment (Muramoto 1975). (2) Preparations were made by the routine flame-drying technique. (3) Air-drying with the preparations in a desicator for 20-24 h. (4) Keeping the preparations in a 0.5 N HC1 solution 216 N. MURAMOTO [Vol. 61(B), for 3-4 h under 20-25°C. (5) Washing the preparations. (6) Air-drying the preparations in a desicator. (7) Staining the preparations in a 40% acetic orcein solution for 2-4 h. (8) Washing the preparations. (9) Air-drying the prepa- rations. (10) Observations. Results. The centromeric regions of some insect chromosomes were clearly denoted by the present method (Figs. 1-4). They were differentially stained Figs. 1-4: Showing centromere regions. 1: Oxya yezoensis (2n, 23. including two plates). 2 : Podisma mikado (MIT, 11. non-X plate). 3 : Aulacophora f emoralis (2n, 22). 4 : Cicadella viridis (MI, 10). The scale A is for Figs. 1-2 and the B is for Figs. 3-4. in the spermatogonial chromosomes of Oxya yezoensis and Aulacophora f emoralis (Figs. 1 and 4), and in the chromosomes of the first or second meiotic metaphase (MI or MIT) of Podisma mikado and Cicadella viridis (Figs. 2 and 3). But the centromeric regions had not been shown by the same method in either spermato- gonial and MI chromosomes of Eurydema rugosa, Palomena angulosa, Lygaeus equestris, Plautia stali, Abraxas sp. and For ficula mikado (Figs. 5-10). The result substantially supports the usual idea concerning a centromere of the insect chromosomes: i.e., colleopteran and orthopteran insects had the normal chromosomes with a single centromere, whereas dermapteran, heterop- teran and lepidopteran insects had the holocentric chromosomes (White 1973). The Hemiptera is taxonomically classified into suborders the Heteroptera and the Homoptera. It has been thought that the chromosomes of the heteropteran and the homopteran insects showed the holocentric nature. Through the present study, four heteropteran species showed a holocentric nature just as expected, but one homopteran species (C. viridis) had not been shown it (Figs. 3 and 5-8). The latter was a new sample for the centromeric nature in the hemipteran chromosomes. No. 51 Holocentric Chromosomes 217 Figs. 5-10: Showing holocentric chromosomes. 5: Eurydema rugosa (2n, 14). 6: Palomena angulosa (2n, 16). 7: Plautia stali (2n, 14). 8: Lygaeus equestris (MI, 8). 9: Abraxas sp. (MI, 28). 10: Forficula milcado (2n, 24). Rithmann and Permantier (1973) proposed a working hypothesis that the distributions of genetic loci for the centromere material in species with the localized and diffuse centromeres were different. Six insect orders (Anoplura, Dermaptera, Hemiptera, Lepidoptera, Mallophaga and Odonata) with the holo- centric chromosomes studied so far take positions on the genealogical tree at random, because the holocentric chromosomes have been found at random among insects orders having no relation with taxonomic genealogy (Fig. 11). This fact would offer an interesting subject for the study of the chromosome evolution of insects. Acknowledgement. The author is very grateful to Emeritus Professor Sajiro Makino, M. J. A., for the improvement of the manuscript and his helpful advice. 218 N. MURAMOTO [Vol. 61(B), Fig. 11. Genealogical tree of the Insecta (Yatsu and Uchida 1972). Species of marked orders have holocentric chromosomes. References Bayreuther, K. 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