152 Proc. Japan Acad., 56, Ser. B (1980) [Vol. 56(B),

30. The Knnetochore of the Le pido ptera. I Chromosomal Features and Behavior in Mitotic and Meiotic-I Cells

By Kodo MAEKI Biological Laboratory, Faculty of Science, Kwansei Gakuin University, Nishinomiya 662

(Communicated by Saj iro MAKINO, M. J. A., March 12, 1980)

To date, holokinetic chromosomes characterized by diffuse kineto- chores have been known to occur in the belonging to the follow- ing orders : Hemiptera (Schrader 1940 ; Ris 1942 ; Hughes-Schrader 1948; Helenius 1952; Rao 1957-58; Halkka 1959; Parshad 1958; Hughes-Schrader and Schrader 1961; Ueshima 1963, 1979) ; Anoplura (Bayreuther 1955) ; Mallophaga (Scholl 1955) ; Dermaptera (Ortiz 1969, White 1971) ; Odonata (Schrader 1947 ; Kiauta 1967-9) ; and in some plants such as Luzula (Nordenskiold 1951, 1961; La Cour 1952; Kusanagi 1962) . The holokinetic chromosomes are generally charac- terized by the behavior with the distinctive nature based on diffuse kinetochore activity. The literature refers to important information on the holokinetic chromosomes with diffuse kinetochore of Lepidop- tera which had been demonstrated by Federley (1943, 1945), and Suomalainen (1953, 1965) on cytological and evolutional bases. On the other hand, White (1973) has denoted in his brilliant book " Cytology and Evolution" that the status of centromeres in the chromosomes of is still somewhat doubtful. The present author has long interested in the structure of the lepidopteran kinetochore, and his work has been continued to inquire into the structure of the kinetochore. Recently the author has obtained some evidence in favor of the holokinetic structure in certain lepidop- teran species. In a series of papers the author wishes to deal with the features and behavior of chromosomes observed in the mitotic and meiotic phases in lepidopteran species with special attention toward the kinetochore. Chromosomal features in the spermatogonial metaphase and anaphase. The following investigations have been undertaken based on the chromosome slides prepared according to the current air-drying squash method. Chromosomes were examined in spermatogonial metaphases and early anaphases in the following 7 butterfly-species : Papilio maackii (2n, 60) ; Papilio bianor (2n, 60) ; Polygonia c-aureum (2n, 62) ; Argyreus hyperbius (2n, 62) ; Curetis acuta (2n, 58) ; No. 3] Kinetochore of Lepidoptera. I 153

Favonius lati f asciatus (2n, 48) ; and Narathura japonica (2n, 48). They are reproduced in Figs. 1-12. The meiotic chromosomes were in-

Figs. 1-12. 1-6: Spermatogonial metaphase. 7-12: Spermatogonial anaphase. 1: Papilio bianor (2n, 60). 2, 6: Curetis acuta (2n, 58). 3: Papilio ma- ackii (2n, 60). 4: Polygonia c-aureum (2n, 62). 5 Argyreus hyperbius (2n, 62). 7 (2 nuclei), 9, 11, 12: Narathura japonica (2n, 48). 8, 10: Favonius latif asciatus (2n, 48). vestigated in six of the above seven species according to the routine paraffin-section method, and their general accounts were already given by the author (Maeki 1953, a, b). Then, the following species, F. lati f asciatus has provided data new to cytology. In general, the mitotic chromosomes have been furnished as 154 K. MAEKI [Vol. 56(B), objects for the determination of chromosomal form in organisms in numerous surveys of karyological studies. As a whole, the mitotic chromosomes provide favorite materials for chromosome analysis from the following reason that the chromosomes of M-I cells are usually presented by a structure of bivalents characterized by their chiasmata. On the basis of the chromosomal aspects derived from the mitotic metaphases observed in the above-mentioned species, the author was not able to obtain any evidence indicating that V- or J-shaped metacen- tric chromosomes are included in the diploid complements of the butterfly. As seen in micrographs of early anaphases shown in Figs. 7 to 12, every chromosome divided each into two elements which took a parallel arrangement. With the advance of the stage these sister chromatids took the start-action to each opposite pole. The chromo- somal features and behavior as seen at the early anaphase were nearly identical to those observed in some Hemiptera such as Eusehistus and Solub ea by Hughes-Schrader and Schrader (1961) . The latter authors have described in their paper that in anaphasic disjunction the separating chromatids remain parallel to each other as they move "broadside on" to the pole . Chromosomal features and behavior in the M-J metaphase and anaphase. The following investigations were undertaken based on the chromosome preparations according to the ordinary aceto-orcein squash method, and also phase contrast was applied for more detailed analysis. Chromosome features depicted at M-I metaphase and ana- phase in the following two species : Graphium sarpedon (n, 20) (butterfly) and variegata (n, 31) (), are presented in Figs. 13-25. The chromosomes of G. sarpedon were reported by Maeki (1957). The accounts from E. variegata are new to cytology. The pre-reductional phenomenon of the bivalents in Lepidoptera had been established on the basis of the behavior of fragment chromo- somes studied through the mitotic and meiotic divisions of G. sarpedon (Maeki and Hayashi, 1979). It was shown that the first division was reductional, whereas the second division was equational. Here, note- worthy was the finding that the bivalent chromosomes of Lepidoptera were constructed by "a ring of four" (La Cour, 1952) at the M-I metaphase (Figs. 13-17, 22-23). The general features of chromo- somes of G. sarpedon and E. variegata at the M-I anaphase are re- markable by the appearance displaying "pseudo-V-shape" in outline see Figs. 18-21, 24-25). In the course of the first anaphase the two sister chromatids moved to the same pole, and then the two chromatids (or a half- bivalent) having the pseudo-V-shape spread gradually along a straight line from each other. It appeared that the spindle fibers attached No. 3] Kinetochore of Lepidoptera. I 155 themselves to the whole poleward surface of the two rods of each sister chromatid. This behavior followed closely the synchronized activity of their spindle fibers. Most reasonable estimation from the above observations seems to be in favor of the view that the formation of the "pseudo-V-shaped" chromosome results from the association of the two rod-shaped ele- ments equipped with the diffuse kinetochore structure.

Figs. 13-25. 13-21: Graphium sarpedon (n, 20). 22-25: Eumeta variegata (n, 31). 13-17: M-I metaphase (a ring of four). 18-2'1: M-I anaphase (pseudo- V-shaped chromosome). 22-23: M-I metaphase (a ring of four). 24-25: M-I anaphase (pseudo-V-shaped chromosome). 13, 14, and 16 following air-drying squash method; others by aceto-orcein squash method. ><2000. 156 K. MAEKI [Vol. 56(B),

Figs. 26-28. Diagram illustrating the morphological change of chromosomes in meiosis. 26: "A ring of four" at M-I metaphase. 27: "Pseudo-V-shaped chromosome" at M-I anaphase. 28: Two sister chromatids arranged on straight line at M-I telophase, and M-II metaphase.

The data presented in this study seem to be supplementary for the information of the holokinetic chromosomes to occur in. Lepidop- tera, particularly presented by Suomalainen (1953, 1965), and Maeki and Hayashi (1979). Further accounts will be presented in a series of papers now in preparation. The author's cordial thanks are due to Emeritus Professor Sa j iro Makino, M. J. A., for revising the manuscript with expert criti- cism, with continuous encouragement.

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