ГЕНЕТИКА, 2014, том 50, № 9, с. 1084-1088
ГЕНЕТИКА ЖИВОТНЫХ
УДК 576.316:599.323.4
A NOTE ON THE PRESENCE OF B CHROMOSOME IN THE SMALL JAPANESE FIELD MOUSE, Apodemus argenteus, IN CENTRAL HONSHU, JAPAN
© 2014 Y. Haga, M. A. Iwasa
Course in Natural Environment Studies, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan e-mail: iwasa.masahiro@nihon-u.ac.jp Received February 11, 2014
Previously, many studies have revealed the presence of B chromosomes in wild mouse taxa of the genus Apodemus (Rodentia, Muridae). In one of the Apodemus species, A. argenteus, which is endemic to Japan, it is known that B chromosomes were confirmed only in individuals (2n = 46 + B chromosome) from Hokkaido, Japan. There is no report of the presence of B chromosomes from other localities in the Japanese Islands. In this study, we analyzed the chromosomal constitutions of 43 individuals of A. argenteus from three localities in Honshu, Japan. A total of three individuals from central Honshu showed 2n = 47, and each individual carried a dot-like B chromosome. In addition, these B chromosome features were analyzed by differential staining methods, and the C- and QM-banding patterns of the B chromosomes were identical to those of the X chromosomal heterochromatic region showing the delayed-fluorescent response. Thus, it is considered that these B chromosomes would be derived from the heterochromatin of the X chromosomes, as reported in previously published papers.
DOI: 10.7868/S001667581407008X
Supernumerary chromosomes (B chromosomes) are well known in mammals, and particularly in species of the genus Apodemus mice, there are many reports of the presence of B chromosomes in A. peninsu-lae, A. agrarius, A. sylvaticus, A. flavicollis, A. mystaci-nus, and A. argenteus [1—17]. Interestingly, in the Apodemus, the number and morphology of the B chromosomes are not stable inter- and/or intra-individual-ly [6, 10, 14]. Therefore, the function and importance of the B chromosomes have been argued from an evolutionary standpoint and remain unclear at present [18-21].
Of the Apodemus species, there are three species in the Japanese Islands, the large Japanese field mice, A. speciosus, the small Japanese field mice, A. argenteus, and the East-Asiatic wood mice, A. peninsulae [22]. The former two are distributed in the Japanese Islands: Hokkaido, Honshu, Shikoku, Kyushu, and neighboring islands, but A. peninsulae is found only in Hokkaido. Previous studies reported that the latter two species carried B chromosomes in the Hokkaido populations [7, 8].
The small Japanese field mouse, A. argenteus, is considered an interesting species cytogenetically because A. argenteus (2n = 46) carries intraspecific chromosomal polymorphisms, such as the presence of XO type [23], the presence of supernumerary chromosomes (B chromosomes) and the delayed fluorescent response by QM- and DA/DAPI-staining at constitutive hetero chromatin regions [8, 24]. Accordingly, some individuals of the Hokkaido population of A. ar-
genteus carried B chromosomes, but none were reported from the other localities [8]. On the basis of the previous study, the B chromosomes of A. argenteus in Hokkaido showed the same staining patterns with the X chromosomal heterochromatin by C-banding, and QM- and DA/DAPI-banding [8]. Thus, it is concluded that the B chromosomes have been derived from the X chromosomal heterochromatin. However, it is not understood why only the Hokkaido population has the B chromosomes in A. argenteus.
Especially considering the frequent properties of B chromosomes in the Apodemus, the presence of them only in the Hokkaido population of A. argenteus is a strange phenomenon. In addition, karyological criteria about A. argenteus have been analyzed by several studies only for specimens from Hokkaido and northern Honshu. Thus, there is no karyological study of A. argenteus samples from other localities. In this study, we reexamined the presence of B chromosomes in A. argenteus samples from Honshu, Japanese Islands.
MATERIALS AND METHODS
A total of 43 individuals of A. argenteus were collected from three localities in Honshu using live traps and were analyzed in this study (table). All the individuals examined are preserved in the author's laboratory as a private specimen collection.
Chromosome preparations were performed from fibroblast cells. For the fibroblast cell samples, we cul-
50 MAI-686 (m) MAI-687 (m) MAI-917 (f)
25
44 45 46 47 44 45 46 47 44 45 46 47 Number of chromosomes (2n)
Fig. 1. Chromosome number distribution in the somatic cells ofthree individuals, MAI-686 (male), 687 (male) and 917 (female), carrying B chromosomes in Apodemus argenteus.
il
XX
Il il II Ii II II I* 1» II "
II • •
Ml M2
II II
— A1
II
— All
- A10
* t
- A20
Fig. 2. A conventionally stained karyotype carrying a B chromosome (specimen No. MAI-917). Autosomes consist of two metacentrics (Ml and M2) and 20 acrocentrics (A1—20). B and X indicate a B chromosome and X chromosomes, respectively.
0
B
tured tail vertebrae tissue in Minimum Essential Medium (MEM; Nissui) including 15% bovine calf serum for 2 weeks in an incubator with 5% CO2. We added colchicine (final concentration 0.025 ^g/mL) at 45 min before harvest. Subsequently, the cells were treated in 0.075 M KCl at 37°C for 20 min as a hypotonic treatment, followed by fixation with Carnoy's fixative (methanol : acetic acid = 3 : 1). Air-dried cells were conventionally stained and differentially stained by QM- and C-banding techniques [25, 26].
RESULTS AND DISCUSSION
Of the 43 mice analyzed, 40 individuals carried the conventionally Giemsa-stained karyotype showing the standard 2n = 46 and FNa = 48 constitutions consisting of 20 acrocentrics and 2 metacentrics of autosomes, and sex chromosomes: a large-sized subtelo-centric X chromosome and a medium-sized acrocentric Y chromosome. In contrast, the other three individuals carried 2n = 47 with a dot-like B chromosome (Fig. 1 and 2). These three individuals were col-
Apodemus argenteus individuals examined in this study
Collection locality Number of individuals B chromosome observed
female male female male
Hirosaki, Aomori Pref. (40°31' N, 140°26' E) - 1 - -
Fujinomiya, Shizuoka Pref. (35°20' N, 138°32' E) 14 20 1 2
Kozagawa, Wakayama Pref. (33°39' N, 135°40' E) 6 2 - -
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Fig. 3. A C-banded metaphase of the Apodemus argenteus individual carrying one B chromosome (specimen No. MAI-917). Arrow and arrowheads indicate a dot-like B chromosome and X chromosomes, respectively.
lected at the foot of Mt. Fuji, Fujinomiya, Shizuoka Pref., central Honshu, Japan (Table). The autosomal and sex chromosomal constitutions were identical to those of individuals with 2n = 46 but there was an extra chromosome in the karyotype.
Previously, the presence of the B chromosomes in A. argenteus was confirmed in only the Hokkaido population [8]. However, the current results indicated that B chromosomes are recognized in specimens not only from Hokkaido but also from Honshu. The present
B chromosomes were quite small and dot-like (Fig. 2); therefore, there is a possibility that these small B chromosomes have been undetected in microscopic observations. Thus, careful observations would lead to discoveries of such B chromosomes.
We counted the number of B chromosomes per 50 cells for the three individuals; the numbers were relatively stable, and those modes of the numbers of diploid chromosome sets were found constantly at 2n = 47 (Fig. 1). Therefore, it is considered that the B chromosomes are equally segregated to daughter cells throughout the somatic cell divisions. Although we did not observe meiotic cell division at spermatogenesis in the present male individuals carrying B chromosomes, meiotic stages may be an important cue to evaluate the meaning of the presence of B chromosomes [27].
To evaluate the origin of these B chromosomes, we performed differential staining for the chromosomes of the three individuals. The C-band patterns were identical to those of the previously published data showing positive C-bands on the autosomal cen-trometic regions, the entire short arm and the proximal region of long arm on the X chromosome and the entire Y chromosome (Fig. 3) [8, 24]. The B chromosomes were also deeply stained by the C-banding (Fig. 3).
We also tried to observe the chromosomes using the fluorescent QM-banding. On the basis of the previously published data [8], the staining behavior of the B chromosomes was fundamentally similar to that in the heterochromatin region of the X chromosome showing delayed-fluorescence behavior, which is specific to A. argenteus [24]. In this result, the B chromosomes tended to have a fluorescent pattern similar to that of the X chromosomal and M2 heterochromatic
»
A %
Fig. 4. Three sequential metaphases with fluorescent QM-banding: initial stage of blue light illumination (a), one minute after blue light illumination (b) and three minutes after blue light illumination (c) (specimen No. MAI-917). Arrows and arrowheads indicate dot-like B chromosomes and X chromosomes, respectively.
TEHETHKA TOM 50 № 9 2014
a
c
regions showing delayed-brightness (Fig. 4). However, because of the smallness of the B chromosome, the fluorescent patterns were sometimes unclear. Namely, the present B chromosomes are probably derived from the X chromosomal and/or M2 heterochromatin, but we were not able to conclude with certainty at present.
ACKNOWLEDGMENTS
We express our gratitude to Dr. Yoshitaka Obara for his encouragement and kind cooperation in collecting mice. Special thanks are also due to Keiki Matsuda and Koichiro Shirai for their kind help with the sampling of mice. This study was supported in part by the International 3-Year Project (2006—2008) ofthe Regional Research Institute ofAgricultural Production, College of Bioresource Sciences, Nihon University,
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