Karyological study on bighead goby ( Neogobius kessleri) from
southern part of the Caspian Sea
Amir Hossein Esmaily - Mohammad Reza Kalbassi*
* Kalbassi_m@modares.ac.ir
Department of Fisheries, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares
University, P.O.Box: 46414-356 Nour, Iran
Abstract
Karyological characteristics of bighead goby (Neogobius kessleri) in the Caspian Sea were
studied by examining 30 metaphase chromosome spreads from the kidney tissue of 10
specimens. The chromosome number of this species was found 2n=46 and the arm number
as NF=46. The prepared karyotype of this species consisted of 23 pairs acro-telocentric (at) chromosomes. The chromosomal formula can be stated as 2n=46 (a-t). Karyological
parameters showed that relative length was between 2.34-7.04 and length variation range of
chromosomes was between 1.67-5.01 and total length was 71.16 µm. It was found that the
best chromosomal spread quality were obtained from intraperitonial injection of 40 µg/g
colchicine for 5 hours, hypotonization of samples in %1 sodium tri-citrate solution in 4ºC
and preparation of spreads on cooled slide with flame technique.
Key words: Chromosome, Karyology, Bighead goby, Neogobius kessleri, Caspian Sea , Iran.
Introduction
Gobies are the most abundant fish in freshwater and oceanic islands. The smallest fishes
(and vertebrates) in the world belong to this family. Mostly are marine fish and found in
shallow coastal waters or around coral reefs. Some species have symbiotic relationships
with invertebrates. Neogobius is found in the Black and Caspian Sea where there are about
11 species, some large enough to be the object of commercial fisheries. The general Persian
name for this genus is gavmahi or sagmahi (Abdoli, 1999).
Systematically, Neogobius kessleri belongs to Actinopterygii class, Perciformes order,
Gobiidae family and Neogobius genus. This endemic fish of the Caspian Sea have been
reported from a wide range of rivers in Iran, from Astara to the Gorgan and probably the
Atrak, Haraz River, Anzali Lagoon and Gorgan Bay as well as the southeast, southwest and
south-central part of the Caspian Sea (Hol ík and Oláh, 1992).
Since the 1960s, karyological studies in teleost fishes have made noteworthy contributions
to increasing knowledge in the fields of genetics, taxonomy and environmental toxicology
(Cucchi & Baruffaldi, 1990). The progress in increasing such knowledge has been closely
related to the evolution of application methodologies (Rivlin et al., 1985). Studies of the
chromosomes of fishes have not been as successful or widespread as in other vertebrate
groups. Standard karyotypes are reported for less than 10% of more than 20000 extant
species of fishes (Gold et al., 1990). The study of fish chromosome has become an active
area of research in recent years (Thorgaard, 1983). Chromosomal analysis is important for
fish breeding from the viewpoint of genetic control, the rapid production of inbred lines,
taxonomy and evolutionary studies (Hosseini and Kalbassi, 2003). Karyological studies
have provided basic information on the number, size and morphology of chromosomes that
is important to undertake chromosome manipulations in fish (khan et al., 2000). Genetic
divergences of populations and their local adaptation are a potential resource for breeding
programs in aquaculture and for fishery management (Philips & Rab, 2001).
Although morphological and anatomical characteristics of this fish has been studied
(Abdoli, 1999), application of non-morphological methods, such as cytogenetics studies,
may provide a framework for the correct species identification of this fish. On the other
hand, due to the less information on Iranian fish karyotypes (Kalbassi and Keyvanshokoh,
2004, Esmaili and Piravar, 2006) the results of this study could be provide the
chromosomes data and karyotype analysis of N. kessleri in Caspian Sea shoreline of Iran.
Materials and methods
Specimens of N. kessleri (n=10, weight=100-150 gr) were caught in Mahmoud-abad shores
of the southern Caspian Sea. The fishes were transported live to our laboratory, and kept in
a well-aerated aquarium at 15 - 20ºC before analysis.
Mitotic inhibitors
The stock solution of colchicine was made by dissolving 10 mg colchicine and 100 mg
NaCl in 20 ml distilled water. The colchicine was administered intraperitoneally at dose of
25 and 40 g/gr body weight. Then, fishes left in aquaria at 15-20˚C for 5-10 hours before
sacrificing. The anterior kidneys were removed after killing the fish and then the
homogenized cell suspension were transferred in a hypotonic solution (0.075M KCl or 1%
sodium tri-citrate) at two different temperatures (4˚C and 25˚C) for about 45-50 min.
Fixation
The swollen cell suspensions were centrifuged at 800 G for 10 min and then fixed in fresh
and cold Carnoy’s fixative solution (3 parts methanol and 1 part glacial acetic acid) for 30
min; then, the old fixative was replaced with the fresh Carnoy’s. Duration of exposure
time for fixation treatment was 60 min.
Spreading
The slides, already washed in alcohol and ether and kept at -1˚C, were prepared by letting
two drops of the fixing solution containing the cell suspension fall onto the cooled slide
with flame and warm slide (40ºC) in different high (60, 90 and 120cm). Thereafter, the
fixative was burned off immediately, using the technique developed by Mellman (1965),
for obtaining better cell spread. The slides were stained in series of concentrations (5, 10
and 15%) of Gimsa Merck solution in distilled water and buffered by phosphate (40 mol
Na2HPO4 and 26.6 mol KH2PO4) at pH 6.8 and were assessed at 7, 8, 9 and 10 min
exposure times to determine optimum staining conditions.
Chromosomal examinations and morphometric measurements
Metaphases were examined under a photomicroscope (Leica SER. No. 990398, Equipped
with a green filter and digital camera). The chromosomes at the metaphase were
photographed with a digital camera (Sony SSC-DC 58 AP) onto Kodak color films (ASA
25). In the course of the microscopic examinations, the chromosomal sets of 30 cells were
counted and 10 of the best mitotic metaphases were used to measure karyotypes. The
morphometric measurements of chromosome pictures were conducted with photographic
software Photoshop 6.0 (Adobe Systems). Each chromosome was tagged with a reference
number. The data were transferred to the Excel 2000 (Microsoft) for analysis.
Chromosome pairing
To increasing distinguishability between the homologous chromosomes, the total length of
chromosome was computed by summing up the average chromatid lengths of each diploid
complement. The length recorded in pixels by the Color Image Analysis System Video Pro
32 (Leading Edge) was converted into micrometers after the scale factor was calibrated
with a stage micrometer.
The chromosome pairs were classified following the recommendations of Macgregor
(1993). The pair numbers and the decreasing length order within each class were definitely
attributed following this classification. Finally, the karyotype was constructed by first
dividing arranging order of the homologous pairs in the decreasing length order within each
group.
Results
Results showed that the number of diploid chromosome in 30 metaphases from the anterior
kidney cells of ten N. kessleri specimens was 2n=46 (Fig. 1). All chromosomes in the
karyotype had a homologous pair, which were arranged in decreasing size. The
investigation of metaphases showed notable difference in size of chromosomes but no
difference between chromosomal type was evident. In addition, the sex chromosomes could
not be distinguished without banding techniques in this species.
The representative karyotype for N. kessleri is shown in Fig. 2. It has 23 pairs of acrotelocentric chromosomes. The number of chromosomal arms was determined as NF=46
and chromosome formula would be expressed as 2n=46 (a-t). The morphological and
numerical data summarized in Tables 1 and 2 show that relative length and length variation
range of chromosomes are between 2.34-7.04 and 1.67-5.01 respectively. Total length of
chromosomes were 71.16 µm. The idiogram of the N. kessleri was made based on the
haploid set of chromosomes (Fig. 3).
In this study, the optimum colchicine concentration for N. kessleri was determined to be 40
g/gr BW of colchicine solution for five hours. This concentration effectively arrested
dividing cells in metaphase stage. In addition, the best chromosomal spread quality (wellspread metaphase) were obtained from treatment of cells with 1% sodium tricitrate solution
at 4oC for 45-50min, while 0.075M KCl, did not result in considerable metaphases.
Table 1- Centromeric index of bighead goby (N. kessleri)
Chromos
ome No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
chromosome
total length (µm)
5.01
4.34
4.01
3.84
3.84
3.51
3.34
3.34
3.17
3.01
3.01
3.01
3.01
2.84
2.84
2.67
2.67
2.67
2.67
2.51
2.51
1.67
1.67
centromer
index
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
arms
ratio
relative
length
7.04
6.09
5.63
5.39
5.39
4.93
4.69
4.69
4.45
4.22
4.22
4.22
4.22
3.99
3.99
3.75
3.75
3.75
3.75
3.52
3.52
2.34
2.34
Chromosomal
type
A*
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
*A: acrocentric
Table 2- Karyotype characteristics of bighead goby (N. kessleri)
Chromosome
number
(2n)
46
Number of
chromosome arms
(NF)
46
Total length of
Chromosome
Haploid total
chromosome length
( µm )
71.16
(µm)
35.58
Figure 1- Karyogram of bighead goby (N.kessleri)
Figure 2- metaphase
Figure 2- Metaphase chromosomes of bighead
goby (N. kessleri) × 1000, 2n=46
Figure 3- Idiogram of bighead goby (N.
kessleri), n=23
Discussion
Several techniques have been developed to examine chromosomes in tissues of adult fish.
These include squashed (Al-Sabti 1983), blood leucocyte culture (Baker, 1972; Al-Sabti,
1985) and cell suspensions from tissues such as gill, kidney and intestine (Kligerman &
Bloom, 1977; Gold et al., 1990), cornea (Drewery, 1964) and scales (Denton & Howell,
1969). Due to previous succesfull results on some species karyotyping in our lab (Hosseini
and Kalbassi, 2003, Kalbassi and Keyvanshokoh, 2004, Kalbassi & Dorafshan, 2005,
Kalbassi et al., 2006) we utilized cell suspension from anterior kidney in present study.
This technique is rather inexpensive and results are obtained relatively fast. Such
techniques are based on the use of Colchicine to block quickly proliferating cell
populations at the metaphase stage. Due to the small size and high number of
chromosomes, karyological study of teleost fishes presents technical difficulties that are not
encountered in the study of other vertebrates (Cucchi & Baruffaldi, 1990).
Karyological study has some different steps. The first step in the procedure is treatment of
the cells with Colchicine, which arrests cell division at metaphase (Baski & Means, 1988).
High concentration and long period of colchicine treatment effect on chromosome, cause to
aggregate and reduce the size of chromosome and their arms, so it is difficult to identify
short arm of an acrocentric chromosomes or also other types of chromosomes. This study
suggests that colchicines concentrations of 50 g/gr BW can effectively arrest dividing
cells in metaphase in kidney tissues. But the maintenance periods may vary according to
species. Also type of hypotonic solutions treatment as well as duration of exposure time,
affect the amount of chromosome spreading. In this study, 0.075 M KCl hypotonic
treatments were ineffective in obtaining well-spread metaphases. Although condensed
chromosomes could be observed, they were often seen inside an intact cell or only slightly
spread. Fixative treatment was not found to be as important as hypotonic treatment in
obtaining well-spread metaphases.
The main difficulty in working with fish chromosomes is in obtaining high quality
metaphase spreads. A few studies have used fish standard karyotypes to examine
taxonomic or systematic problems (Bolla, 1987). The major difficulty encountered is the
morphological variation existing even between homologous chromosomes in the same
nucleus (Al-Sabti, 1991 & Levan et al., 1964). Sometimes it could happen that some
chromosomes are more contracted than others, so chromosome measurements are very
difficult in fishes which have small chromosomes compared to those of man and mammals.
Another problem is that fish karyotypes are not identical as in human being or other animal
species, so we can not have a standard karyotype for fish because not only are there
differences between species, but polymorphism often occurs within the same fish species
(Al-Sabti, 1991). Several incomplete metaphases were encountered in the preparation, and
these probably resulted from hypotonic over treatment (Nanda et al., 1995). The majority
of authors classify uni-armed and bi-armed chromosomes according to the guidelines of
Macgregor (1993).The majority of Gobiidae species have 2n = 46 chromosomes while
Neogobius fluviatilis and Neogobius melanostomum have 2n = 42-46 (Klinkhardt, et al.
1995). Until now, karyotype of some members of Neogobius genera was determined like as
Neogobius melanostomus affinis (2n=46, NF=46 2n=46a-t) (Klinkhardt, et al. 1995).
Additional data from this species and related taxa may provide beneficial insights into the
value of conventional cytogenetic data for reconstructing Gobiidae family. However, the
value of karyological data can be better utilized if combined with the highest possible
taxonomic elements for the diagnosis of species. In addition, the karyotype analysis is a
key step toward the stock improvement by polyploidy manipulation, hybridization and
related genetic engineering (Tan et al., 2004). Therefore, like other animals, comprehensive
genetic researches is needed for this fish as well.
Acknowledgment
We thank Mr. Ahmad zadeh from Khoram Company for kindly providing N. kessleri as
well as Mr Kamali for technical assistance.
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