Karyotype analysis of the red swamp crayfish, Procambarus clarkii, by cell culture

1 Pages·1991·0.34 MB·

98 MEMOIRS OF THE QUEENSLAND MUSEUM
KARYOTYPE ANALYSIS OF THE RED Ui ay 0p Ce ee re ah ee at
SWAMP CRAYFISH, PROCAMBARU'S
th we dy ¢4 we vt re ab Gh at
CLARKII, BY CELL CULTURE
Karyological analysis of the red swamp crayfish. Procam- ap ero re re a8 cf ve 48 08 08
barus clarkti, was examined using a cell culturing technique. aeoga tp we tt at v. Hs cc re
The specimens used for the present study were collected
we-ur ve 02 re ef so sa oF an
from a rice field near Mishima City, Shizuoka Prefecture,
Japan. we
The procedures of the cell culture and chromosome pre-
parations were as follows, The bodies of the specimens were
sterilized with 70% ethanol before the tissues were taken out,
The antennal and genital glands were dissected out asepti-
cally and cul into 1-2 mm pieces, The pieces of tissue were
immersed
in calcium- and magnesium- free Hank's solution tor 20
minutes, and then in collagenase solution for 1-3 hours. The
dissociated cells and smal! clumps of cells were collected by
centrifuge (1,000 rpm for 5 minutes) and then washed in PC
FIG,], Karyotype of the red swamp crayfish, Procambarus
culture medium (Table 1). The collected cells. and small
clarkii.
clumps of cells were put into plastic tissue culturing petri
The increased cells were removed by wypsin treatment and
dishes with new PC medium at 26°C, After the epithelial and
treated with hypotonic solution (0.01 M KC1) for 6-12
fibroblastic cells increased sufficiently (3-7 days), colchi-
minutes, The cells were fixed in fresh ethanol and acetic acid
cine (0.5 g/ml) was added to the medium for 6-15 hours.
solution (3:1), The chromosome preparation was modified
from our routine air-drying method for Crustacean chromo-
TABLE 1. Composition of the culture medium (PC). some preparations (Murofushi and Deguchi, 1983).
The diploid chromosome number of P. clarkii was 188
Amino Acids (me/)| Salts (2n) with considerable variation in size as was found by
L-Arginine.HCl ; Nac Murofushi er af. (1984). The chromosomes were classed as
L-Aspartic acid A KC] 63 pairs of meta-, 10 pairs of submeta-, 7 pairs of subtelo-
L-Asparigine,H20 MgSQ47H30 and 14 pairs of acrocentrics (Fig. 1), The metacentric chro-
L-Aranin MegC126H20 mosome numbers formed 67%, and others were 15% inacro-,
B-Aranin CaCis2H70 10% in submeta- and 8% in subtelocentrics, The culture
L-Cystin method provided more cells in milofic metaphase than direct
L-Glutamic acid lreatment with colchicine, The chromosomes were also more
L-Glutamin Sugars distinct.
Clycine Clucose
L-Histidine Fructose Literature Cited
L-lsoleucine Saccharose Murofushi, Mand Deguchi, Y. 1983. A method for obtaining
L-Leucine mclaphase chromosomes from large shrimp-like
L-Lysin.HCl crustaceans, Report of the Mishima Rescarch Institute
L-Menhonin Buller (g/l) of Sciences for Living, Nihon University 6: 31-34,
L-Proline HEPES 3.5745 Murofushi, M., Deguchi, Y and Yosida, T.H. 1984, Karyo-
L-Phenilalanin logical study of the red swamp crayfish and the Japanese
DL-Serin
lobster by air-drying method. Proceeding of Japan
L-Tyrosin
L-Trypltophane Antibiotics Academy 60, Ser. B: 306-309.
L-Threoninc Gentamycin = L00ug/ml
M,. Murofushi, Junior College at Mishima, Nihon
L-Vualine
University, Mishima, 411 Japan.
L-Cystein. HC] Scrum
L-Hydroxyproline 10% Fetal Bovin Serum
T. Nakatsubo and Y, Deguchi, Department af
Fisheries, Nthon University, Setagaya, [54 Japan.