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EXPANSION OF CYTOGENETICALLY MODIFIED FORM OF OPISTHORCHIS FELINEUS IN THE TOM RIVER BASIN
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EXPANSION OF CYTOGENETICALLY MODIFIED FORM OF OPISTHORCHIS FELINEUS IN THE TOM RIVER BASIN
Ilyinskikh Nikolai 1
Ilyinskikh Ekaterina 2
Authors:
1.  National Research Tomsk State University

Russian Federation
2.  Siberian State Medical University

Russian Federation
Type:
Article
DOI:
https://doi.org/10.21603/2500-1418-2016-1-2-65-79
Pages:
from 65 to 69
Status:
Published
Received:
30.12.2016
Accepted:
30.12.2016
Published:
30.12.2016
Subject area:
GRNTI 29.01 Общие вопросы физики
GRNTI 27.01 Общие вопросы математики
GRNTI 31.01 Общие вопросы химии
GRNTI 34.01 Общие вопросы биологии
Language:
English
Keywords:
Opisthorchis felineus, karyotype, Robertsonian translocation, Tom River
Abstract and keywords
Abstract (English):
In 1993, karyotyping of Opisthorchis felineus sporocysts, isolated from aquatic mollusks Bithynia inflat a in the industrial wastewater of the Siberian Chemical Combine discovered cytogenetically modified 12-chromosome specimens. In 2012-2013, a repeated study of the spread of these mutant trematodes in the lower course of the river Tom was carried out. It was established that specimens with sporocysts of O. felineus having 2n=12 were found in 8 of 10 places of catching. The caught goldfish were used to isolate metacercariae to infest golden hamsters in the experimental conditions. After 5 months, the hamster livers were processed to extract marita specimens and the number of chromosomes in their ovaries and testes was cytogenetically determined. The results of analysis of the spread of O. felineus carried out by this method showed the presence of 12-chromosome form of the Opisthorchis marita in all places of fishing out in the lower course of the river Tom. Analysis of the 12-chromosome karyotype allows to conclude that this mutation was a result of Robertsonian fusion of chromosomes 3 and 6 of 14-chromosome form of karyotype.

Keywords:
Opisthorchis felineus, karyotype, Robertsonian translocation, Tom River
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INTRODUCTION Opisthorchis felineus (Rivolta, 1882) trematode of Opisthorchiidae family (Braun, 1901) parasitizing at various developmental stages in aquatic mollusks of Bithyniidae family, fish of Cyprinidae family and in piscivorous mammals, including humans. Karyotype analysis of this species was carried out by Ya.V. Barshene [1], who established that 2n = 14 (4m+6sm+4a); similar results were obtained by our studies [2, 3]. In the study of Polyakov et al. [4], diploid set of chromosomes was 2n = 14, too, but the ratio of morphological types of chromosomes was somewhat different (6m + 8sm). As a result of accident at the Siberian Chemical Combine in 1993, a significant number of radionuclides was released into the environment, contaminating local water bodies, which caused accumulation of long- lived and short-lived radioactive elements in aquatic organisms [5-7]. Chromosome analysis of sporocysts of O. felineus, extracted from Bithynia inflata aquatic mollusks caught in SCC technological effluents, allowed to establish a significant increase in the number of trematode cells with chromosomal aberrations, as well as change in the number of chromosomes. [3] As it turned out, in one of the local SCC waste discharge sites more than 30% opistorhides had balanced reciprocal Robertsonian translocation with emergence of a couple of large submetacentric chromosomes. Karyotype formula of these cat liver flukes was 2n = 12 (4m + 8sm). The main objective of this research was to study again, 20 years after the incident, cytogenetic consequences of radioactive pollution for the population of O. felineus and to solve the problem of the further spread of the cat liver fluke with Robertsonian translocation the lower course of the river Tom (Russian Federation). MATERIALS AND METHODS During the summer period of 2012-2013, 124 specimens of Carassius auratus gibelio goldfish were caught. This species is one of the most frequently encountered in catches at all points of the lower course of the river Tom, and the only species of fish present in catches from the neighboring waste discharge site of the Siberian Chemical Combine. Mature fish were caught in 10 places of the lower course of the river Tom and its tributary, the river Romashka (Fig. 1). Metacercariae were isolated from fish muscles with the use of the method described in the study of D.P. Kudryavtsev [8]. Invasion of 120 golden hamsters by giving 30-50 metacercariae of O. felineus into a throat. After 50 days, Opisthorchis marita specimens were isolated from the liver of hamsters at the prosection. Chromosomal preparations from marita specimens Please cite this article in press as: Ilyinskikh N.N., Ilyinskikh E.N. Expansion of cytogenetically modified form of Opisthorchis felineus in the Tom river basin. Science Evolution, 2016, vol. 1, no. 2, pp. 65-79. doi: 2500-1418-2016-1-2-65-79. Copyright © 2016, KemSU. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. This article is published with open access at http:// science-evolution.ru/ were prepared by the method of Terasaki [9] with some modifications. Live marita specimens of O. felineus were placed in 0.005% colchicine physiological saline solution for 3 hours at 37°. Then their testes and ovaries were removed and placed in 0.6% hypotonic sodium citrate solution. Then cells were fixed for 30 minutes in a Carnoy’s solution (methanol + glacial acetic acid in the ratio of 1: 1). Furthermore, a drop of cellsuspension was placed on a chilled wet glass and dried by hot air flow with a hair dryer. Ob River Tom River Fig. 1. Schematic map of places of catching Carassius auratus gibelio goldfish and Bithynia inflata mollusks in the Tom river basin. Places of catching fish and mollusks: 0 - SCC waste settling ponds at the river Romashka; - Romashka river mouth; 2 - near the village of Chernilschikovo; 3 - near the settlement of Samus; 4 - near the village of Kizhirovo; 5 - near the village of Orlovka; 6 - Labaznaya branch; 7 - Bitenevskaya branch; 8 - Tom mouth; 9 - Chatskaya branch; 10 - near the village of Nagornny Ishtan. In another series of studies, chromosome preparations were obtained from sporocysts of O. felineus isolated from Bithynia inflata aquatic mollusks - one of the main intermediate hosts of O. felineus in West Siberia [10]. 864 mollusk specimens were caught at the bottom of the river Tom and its tributary the river Romashka. Examination of these mollusks showed that in 53.7-94.5% of cases, depending on the catching location, they were infested by the larvae of O. felineus. Species belonging to O. felineus was determined by cercariae isolated from infested mollusks. For chromosomal preparations of O. felineus, caught mollusks were placed in 0.005% colchicine solution for 12 hours. Water for the solution was taken from the river. Chromosomes preparations were prepared of sporocysts. For this purpose, the isolated larvae were treated with colchicine and placed for 1 hour in distilled water, which was changed three times. Fixation was performed in Carnoy’s mixture (3 parts of ethanol per one part of glacial acetic acid). In all cases, the preparations were stained according to Romanovsky-Giemsa method with the use of Sorensen’s phosphate buffer (pH 6.8) diluted at a ratio of 1:50 for 15 min. The chromosome study was conducted with the use Reichert NP-1640 microscope (Austria) and 10 × 90 magnification. The karyotyping used classification of chromosome types proposed by Ya.V. Barshene [1] and updated by A.V. Polyakov et al. [4]. RESULTS AND DISCUSSION Our findings, as well as results of the other research [1, 4, 11] indicate that the karyotype of O. felineus normally has 14 chromosomes. From our point of view, the most interesting of them is our observation of emergence and spread of specimens of O. felineus with 12-chromosome karyotype as a result of Robertsonian translocation of acrocentric chromosomes (Fig. 2). Fig. 2. Karyotypic features of Opisthorchis felineus with 2n=12 and 2n=14 According to karyological data, emergence of 12- chromosome variant is associated with centric fusion of 3 and 6 chromosomes of 14-chromosome karyotype of O. felineus. The results of this study suggest a significant spread of a modified form of O. felineus in the lower course of the river Tom for the period from 1993 to 2013 (Table 1). Analysis of incidence of 12-chromosome forms of sporocysts of cat liver flukes isolated from Bithynia inflata snails shows that in 1993 the main trematode spread areas were the river Romashka and stretch of the river Tom near the village of Chernilschikovo. 12-chromosome variant of Opisthorchis was not found downstream. Catching of snails in 2013 discovered occurrence of a mutant form of Opisthorchis sporocysts in the river Tom, up to the mouth. Analysis of O. felineus marita chromosomes (Table 2) derived by artificial infestion of golden hamsters by Table 1. Comparative incidence of intact and mutant forms of O. felineus sporocysts in the lower course of the river Tom in 1993 and 2013 Snails catching places Bithynia inflata Number of O. felineus sporocysts 2n=14 2n=12 In 1993 In 2013 In 1993 In 2013 abs. % abs. % abs. % abs. % 0 - SCC waste settling ponds 29 61.7 48 66.7 18 37.3 24 33.3 1 - Romashka river mouth 34 73.9 36 66.7 12 26.1 18 33.3 2 - near the village of Chernilschikovo 23 69.7 24 55.8 10 30.3 19 44.2 3 - near the settlement of Samus 20 100 39 58.2 0 0 28 41.8 4 - near the village of Kizhirovo 41 100 42 95.5 0 0 2 4.5 5 - near the village of Orlovka 25 100 32 88.9 0 0 4 11.1 6 - Labaznaya branch 20 100 14 82.4 0 0 3 7.3 7 - Bitenevskaya branch 32 100 26 86.7 0 0 4 3.3 8 - Tom river mouth 34 100 24 92.3 0 0 2 7.7 9 - Chatskaya branch 30 100 13 100 0 0 0 0 10 - near the village of Nagorny Ishtan 31 100 22 100 0 0 0 0 Table 2. Incidence of intact and mutant marita forms of O. felineus in the lower course of the river Tom Orfe catching places Number of marita specimens studied (abs.) Number of marita O. felineus 2n=14 2n=12 abs. % abs. % 0 - SCC waste settling ponds 34 29 85.3 5 14.7 1 - Romashka river mouth 42 34 81.0 8 19.0 2 - near the village of Chernilschikovo 32 23 71.9 9 28.1 3 - near the settlement of Samus 28 20 71.4 8 28.6 4 - near the village of Kizhirovo 41 41 90.9 4 9.1 5 - near the village of Orlovka 29 25 86.2 4 13.8 6 - Labaznaya branch 28 20 71.4 8 28.6 7 - Bitenevskaya branch 34 32 94.1 2 5.9 8 - Tom river mouth 42 34 81.0 8 19.0 9 - Chatskaya branch 36 30 83.3 6 16.7 10 - near the village of Nagorny Ishtan 38 31 81.6 7 18.4 metacercariae of the parasite isolated from the caught goldfish showed that spread of this trematode is much wider and reached Chatskaya branch and Nagorny Ishtan, which are directly connected with the river Ob (Fig. 1). It is shown that opistorhide larvae are very sensitive to radiation that even allows authors [12] to recommend to decontaminate affected metacercariae fish by irradiation. Comparative analysis of chromosome numbers in trematodes showed a tendency to a decrease in the number of chromosomes in evolution of their karyotype [1, 13]. Some papers proposed karyotype with 19 pairs of acrocentric chromosomes as the ancestral karyotype of trematodes. S. Ono [14] believes that evolutionary transformations often proceed by reducing the number of chromosomes in the karyotype due to Robertsonian translocations. Some scientists [13, 15] also suggest that evolution of the trematode karyotype form proceeds in the form of centric mergers. A significant number of 12-chromosome trematodes of O. felineus in the lower course of the river Tom speaks in favor of the assumption that these species in the ecologically unfavorable situation have some advantages over the normal 14-chromosome O. felineus. There is an opinion that species having a large number of chromosomes are more radiosensitive in the karyotype [16, 17]. According to A.V. Toropov [5], 9 γ-emitting man-made radionuclides were found in the ichthyofauna objects of the lower course of the river Tom. At the same time, 65Zn has the highest absolute level (1650 Bq/kg) and the highest factor of build-up in fish organs and tissues in relation to water. Goldfish (Carassius auratus gibelio) accumulates man-made radionuclides more than other representatives of the ichthyofauna. Besides the rapid spread of 12-chromosome species is due, apparently, to the fact that cat liver flukes in larval development period multiply rapidly by parthenogenesis, and sporocysts give rediae, and rediae give the next stage of larval development - cercariae, in which case one sporocyst may parthenogenetically produce several thousands of descendants [2, 10]. Therefore, the rapid spread of mutant forms of the parasite is possible. It is known that among 5 species of Opisthorchiidae family (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875), only one species, O. viverini, has 12-chromosome karyotype [18]. It was found that the chromosome 3 of Opisthorchis viverrini occurred during the merger of chromosomes 3 and 7 of a possible ancestral karyotype of opistorhides [19]. We do not exclude the possibility of “introduction” of O. viverini in the river Tom, but in this case, it is difficult to explain the fact that it occurred in the radioactive contamination zone, but not in an environmentally safe part of the reservoir. In this regard, we tend to believe that long-term radiation effects on the population of O. felineus in the radioactive waste dumping area may have led to occurrence and spread of 12-chromosome species of this parasite. It is possible that we are dealing with a new kind of opisthorchide flukes. It is known that Opisthorchis group includes a several dozen of species. Some scientists believe that species of O. felineus may be divided taxonomically into separate subspecies. [10]. The same is evidenced by differences in clinical manifestations of the humans opisthorchiasis observed in the Yenisei and Ob-Irtysh regions of Siberia [20]. In this connection, it is of interest to compare the morphology, development cycles, and degree of pathogenicity for intermediate and main hosts, including those for human of 14 and 12-chromosome variants of O. felineus in order to determine degree of taxonomic independence of these groups of trematodes. CONCLUSION In 1993, karyotyping of Opisthorchis felineus sporocysts, isolated from aquatic mollusks Bithynia inflatа in the industrial wastewater of the Siberian Chemical Combine discovered cytogenetically modified 12-chromosome specimens. In 2012-2013, a repeated study of the spread of these mutant trematodes in the lower course of the river Tom was carried out. It was established that specimens with sporocysts of O. felineus having 2n=12 were found in 8 of 10 places of catching. The caught goldfish were used to isolate metacercariae to infest golden hamsters in the experimental conditions. After 5 months, the hamster livers were processed to extract marita and the number of chromosomes in their ovaries and testes was cytogenetically determined. The results of analysis of the spread of O. felineus carried out by this method showed the presence of 12-chromosome form of the Opisthorchis marita in all places of fishing out in the lower course of the river Tom. Analysis of the 12-chromosome karyotype allows to conclude that this mutation was a result of Robertsonian fusion of chromosomes 3 and 6 of 14-chromosome form of karyotype. ACKNOWLEDGEMENT This study was supported financially by grant no. 10190 of the Russian Humanitarian Science Foundation and grant no. 16-44-700149 of the Russian Foundation for Basic Research.
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