Modified Method of Metaphase Plates Obtaining for Polyploid Fish Genera Carassius and Cobitis Karyotyping (Actinopterygii, Cypriniformes)

Abstract Modern methods for obtaining metaphase plates from the somatic cells of fish, most effective in the study of polyploid species, were selected and tested. The technique with CoCl2 and colchicine treatment is recommended on the basis of empirical data for representatives of the genera Carassius and Cobitis. Th e detailed description of this modified technique and characteristics of metaphase plates of Carassius auratus Linnaeus, 1758, Carassius carassius Linnaeus, 1758 and triploid form of Cobitis taenia Linnaeus, 1758 received under tested method is presented in the paper. Abstract Ото- браны и апробированы современные методики получения метафазных пластинок соматических клеток рыб, дающих наибольший эффект при исследовании полиплоидных видов. На основании эмпирических данных в отношении представителей родов Carassius и Cobitis рекомендуется ме- тодика с использованием CoCl2 и колхицина. В статье приведено подробное описание модифи- цированной методики и полученные в результате её применения метафазные пластинки Carassius auratus Linnaeus, 1758, Carassius carassius Linnaeus, 1758 и триплоидной формы Cobitis taenia Linnaeus, 1758.


Introduction
Two basic methodical approaches: direct (in vivo) and indirect (in vitro), are currently applied for obtaining mitotic metaphase of animals. Th e fi rst approach is rather simple, inexpensive and allows quick result. It is associated with the use of colchicine that blocks proliferative cells of kidneys, spleen, gill epithelium, etc. at the metaphase (Kligherman, Bloom, 1977;Hartley, Horne, 1983, and others). Main disadvantage of this approach is a small number of metaphases produced in preparations.
Indirect approach involves prior cultivation of isolated organism's cells on the growth media in vitro (Gold, 1979;Orlov, Bulatova, 1983;Hartley, Horne, 1983;MacGregor, Varley, 1986, and others). This sort of techniques makes it possible to get much better results, as in such a case the stimulators of mitosis, like phytohemagglutinin (PHA) and fetal calf serum (FCS), are applied (Hartley, Horne, 1983). However, this approach requires special equipment, high quality serum and PHA, takes more time than the direct approaches, and is related with a risk of bacterial infection. Besides, changes in the number and morphology of chromosomes may take place in cell cultures, especially in long-term ones (Amemiya et al., 1984).
Application of the stimulators of mitosis in the direct approaches could be regarded as a compromise. Th at sometimes enables to substantially improve the quality and increase the number of metaphase plates produced in preparations (MacGregor, Varley, 1986;Manilo, 1986).
Contrary to the intensive development of cytogenetic investigations in different groups of the vertebrates during last decade, such studies of fish were not so successful and large-scale (Hartley, Horne, 1985). The reason apparently is relatively large number of small-sized chromosomes in their diploid sets (Gold, 1979).
Attempts to use the tissues of embryos (Simon, 1963), as well as juvenile and mature individuals: like epithelium of scales and gills, anterior kidney, testis, heart tissues (Lakra, Bhondae, 1996;Völker, Kullmann, 2006;Kalous et al., 2010, and others), were carried out as to improve existing techniques for producing of karyological preparations of fi sh.
Cytogenetic studies of fi sh are developing intensively during recent decades due to gradual improvements in the techniques for obtaining chromosomal preparations. Mitosis stimulants, such as phytohemagglutinin (PHA), are commonly used for fi sh injection before colchicine treatment (Lin, 1982;Gold et al., 1990;Baruff aldi et al., 1992). Th is substance can induce synthesis of the chromosomal DNA and transition of cells that were nonproliferating in norm, into the mitotic cycle of division. Mitogen of American Pokeweed, that derived from the roots of Phytolacca americana (Fames et al., 1964) and is similar to the PHA, is applied quite frequently. Th at mitogen is characterized by low leuco-agglutinative properties, and is particularly highly eff ective in cases when the PHA does not allow achieving necessary results. Good potential for cytogenetic study of fi sh has application of phenylhydrazine (Cucchi, Baruff aldi, 1988), horse serum (Ojima, Kurishita, 1980), cobalt chloride (Cucchi, Baruff aldi, 1990), concavalin A (Banerjee, 1987) and yeasts (Oliveira et al., 1988).
At the same time, current techniques for producing karyological preparations of fi sh are not always suffi ciently rewarding and require some improvements, especially regarding polyploid species. Th e number of chromosome preparations with high quality metaphase plates is infl uenced by such factors as the method of fi shery and period of specimen delivery to the laboratory, experimental conditions, research equipment, season, etc.
Th erefore, objective of the study is the selection and modifi cation of most productive and available techniques for obtaining metaphase plates of fi sh in our conditions that would be relevant for further analysis.

Material and methods
Th e material for the study were specimens of spined loaches and crucian carps fi shed out in six diff erent water reservoirs of Zhytomyr oblast, Ukraine (table 1) during June and July, 2013. Examined localities are represented by rivers, forest lakes, canals and marshes.
Most eff ective one for the study of Osteichthyes genera Cobitis and Carassius proved to be our modifi ed technique, based on air-drying of slides (Ráb, Roth, 1988;Cucchi, Baruff aldi, 1990;Boroñ, 1994). It is based on usage of cobalt chloride solution, as a stimulator of mitosis, and colchicine solution for working with kidneys.
Th e technique that has been applied for obtaining metaphase plates is rather multistep, thus it is described successively.
P r e l i m i n a r y t r e a t m e n t. Cobalt chloride at the rate of 1 ml of a CoCl 2 / per 100 g body mass (0.1 % solution for crucian carps and 0.03 % solution for spined loach was administered intraperitoneal; then fi sh were placed into well-aerated tanks for one day. Aft er 24 h, 0.1 % solution of colchicine at the rate of 1 ml / per 100 g body mass was applied. Aft er 1.5-2 h, the following procedures were carried out. K i d n e y i s o l a t i o n a n d p r e p a r a t i o n o f s u s p e n s i o n. Widest part of the kidney was removed from fi sh body, aft er that it was dissected by microscopic needles with adding of 3-4 drops of hypotonic solution up to formation of homogeneous suspension H y p o t o n i z a t i o n a n d p r e l i m i n a r y f i x a t i o n. Using a syringe, cell suspension was placed into centrifuge tubes with 5 ml of a 0.075 M KCl hypotonic solution. Hypotonic treatment was performed in an incubator at 37 o C for 25-40 min; period of treatment was determined empirically for diff erent species of fi sh. Aft er hypotonization, 5 drops of freshly prepared cold fi xative (3 methanol: 1 acetic acid) were added to the

T a b l e 1. Materials used in karyologycal analysis
Т а б л и ц а 1. Материал, использованный для кариотипического анализа tubes and suspension was gently stirred with a Pasteur pipette. Later, 5 ml of fi xative were added in 5 min, and then the suspension was stirred once more. C e n t r i f u g a t i o n a n d f i x a t i o n. Final suspension was centrifuged at 1300 rpm for 10 min. Supernatant then was gently removed and 5 ml of freshly prepared cold fi xative was added and stirred. Th e suspension was left at 4 o C for 25 min, and then was centrifuged. Th e last procedure was repeated 3 times. Totally, centrifugation was fulfi lled 4-times and general period of fi xation was at least 2 h. S l i d e s p r e p a r a t i o n. Aft er the last fi xation, supernatant was removed, a few drops of fi xative were added to the cell suspension, all gently stirred and 3-4 drops of the concentrated cell suspension were dropped onto cold, wet slides from a distance of about 10-30 cm. Th ese slides were dried over fi re or under an electric lamp, washed in ethanol and dried again. Th e slides were stained in azure-eosin solution by Romanovsky (1891), for 15 min.
Metaphase plates were analyzed and their photographs were taken under the light microscope Delta Optical Genetic Pro. Th e slides with most distinct morphology of chromosomes that were used for karyograms and further comparative karyological study were selected for taking pictures. Th e chromosome forms were defi ned according to classifi cation by Levan and others (Levan et al., 1964).

Results and discussion
Investigations were based on two diploid species of the genus Carassius, which are ancient amfi diploids (Vasiliev, 1985), and triploid representatives of Cobitis taenia Linnaeus, 1758. Studied species of the genus Carassius were C. auratus Linnaeus, 1758 and C. carassius Linnaeus, 1758, diagnosed by biochemical gene marking.
Application of proposed modifi ed technique for producing chromosomal preparations resulted in obtaining metaphase plates (fi g. 1) suitable for further analysis. Received results are presented in . About a hundred metaphase plates of each species in the whole were analyzed. Under the execution of all technical requirements of the method, the percent of slides with metaphase plates verged towards 90 % and the number of plates applicable for morphological analysis was 60 %. Such result is quite successful, since provides with adequate amount of material for analysis and allows realization of complete cytogenetic study in diploid-polyploid complex of fi sh.
Karyotyping is the most reliable method to check the ploidy. This is direct method, based on counting the number of metaphase chromosomes in somatic cells. Due to the fact that kidneys of fish have hematopoietic function and their cells are regularly dividing, this organ is most suitable for kariological study and assessment of ploidy level.
Treatment by a solution of colchicine is the standard procedure for majority of current cytogenetic techniques applied to fi sh. Th is chemical can block mitotic division of a cell during the metaphase. Usage of cobalt chloride was revealed to be eff ective for stimulation of cell division. Cobalt chloride stimulates formation of erythropoietin, leading to increase of proliferative cells in hematopoietic organs (Webb, 1962).
Optimal concentrations of cobalt chloride were applied for producing the karyologic preparations; those concentrations had not resulted in the death of the examined fish during preliminary treatment and had assisted to obtain high quality metaphase plates.

Conclusions
Th e applied technique for producing the preparations of mitotic chromosomes was eff ective for diploid-polyploid complexes of fi sh. Its application provided an opportunity, in a relatively short period of time, of obtaining metaphase plates that were suitable for further quantitative and qualitative analysis.
Th ese obtained metaphase plates are characterized by sharp boundaries; individual chromosomes are clearly identifi ed and densely arranged within the plate; the number of overlapping chromosomes is minor and that allows karyotyping and assessment of chromosomes' morphological types.
A B C the morphological analysis was carried out. This technique with minor modifications can also be successfully tested on other representatives of freshwater and salt water fish taxa.