ISSN 0095-4527, Cytology and Genetics, 2008, Vol. 42, No. 4, pp. 267–271. © Allerton Press, Inc., 2008.
Original Russian Text © I.A. Kozeretskaya, A.V. Protsenko, E.S. Afanas’eva, S.R. Rushkovskii, A.I. Chuba, T.A. Mousseau, A.P. Meller, 2008, published in Tsitologiya i Genetika,
2008, Vol. 42, No. 4, pp. 63–68.

Mutation Processes in Natural Populationsof Drosophila melanogaster and Hirundo rusticafrom Radiation-Contaminated Regions of Ukraine
I. A. Kozeretskayaa, A. V. Protsenkoa, E. S. Afanas’evaa, S. R. Rushkovskiia,
A. I. Chubaa, T. A. Mousseaub, and A. P. Mellerc
aTaras Shevchenko National University, Kyiv, Ukraine

e-mail: kozer@mail.univ.kiev.ua
bUniversity of South Carolina, Columbia, South Carolina, USA
cPierre and Marie Curie University, Paris, France

Received June 13, 2007

Abstract—Natural populations of Drosophila melanogaster and Hirundo rustica from regions of Ukraine
exhibiting different levels of radiation contamination are investigated. Genetic monitoring was performed withrespect to such parameters as the frequency of visible sex-linked mutations, frequency of gonad reduction in
Drosophila, and rate of interphase manifestations of chromosomal instability in the erythrocytes of the birds.
The results attest to a possible opposite dependence of the level of chromosomal instability among swallows
and that of the rate of lethal mutations in the sex chromosome of Drosophila on the density of radiation contamination.
DOI: 10.3103/S0095452708040099

INTRODUCTION
More than 20 years have passed since the accident at
the Chernobyl nuclear power plant. An enormous quantity of scientific studies have been carried out in thecourse of these years by scientists from different countries along the most varied directions for the purpose ofanswering a question of the greatest importance formankind: What are the consequences of this extraordinary event? The official position of the United Nations[1] is that the consequences of the accident for humanhealth have turned out to be significantly less than
expected. An increase in the animal population has
been observed in the exclusion zone of the Chernobyl
nuclear power plant and this was interpreted as a reduction of the natural ecosystems. Such opinions arose forseveral reasons: (1) Most studies have been performed
under laboratory conditions, which means that theirresults cannot be simply translated to populations processes, hence for precisely this reason they were
not included in the reports of governmental organizations [2]. (2) Classical model subjects were not madesufficient use of in radiobiological investigations,whereas it is only with the use of exhaustively studied
species of living organisms that it is possible to obtainreliable results (mice constitute an exception here [3]).
(3) The results of scientific studies are often contradictory, which may be explained by the features of thestudy subjects and the diversity of the techniques
employed [4].

Vast regions of the territory of Russia, Ukraine, andBelarus as well as other European countries wereaffected by contamination produced by long-lived
radioactive isotopes as a consequence of the accident at
the Chernobyl nuclear power plant. This means thatmost of the animal and plant populations as well as thehuman population are living under conditions ofchronic low-dose radioactive contamination, thegenetic consequences of which have not been studied atall, while the results for those species which have beenstudied have been contradictory.
The objective of the present study was to investigate
mutational processes in natural populations of modelorganisms (Drosophila melanogaster and Hirundo rustica) that reflect the genetic consequences of chronic
low-dose irradiation in conjunction with other factorsof environmental pollution.
MATERIALS AND METHODS
Drosophila melanogaster is a “cosmopolitan” species commonly present at sites where subject matter iscollected as well as a classical model subject. Under
laboratory conditions the fly is bred in a standard
medium [5]. At optimal temperatures, the length ofdevelopment of an individual, from the egg phase to theimago, is roughly two weeks.
The forest swallow, Hirundo rustica, a small bird(≈20 g) from the order Passeriformes, feeds on insects,
principally Diptera and Hymenoptera, which are cap-

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tured in the summer. The males and females constructnests on (or within) man-made structures and thefemales usually lay 4 or 5 eggs. More than half thefemales may lay a second clutch of eggs. Forest swallows live in isolation or in colonies that may exceed
120 pairs. The forest swallows of Ukraine winter insouthern Africa, where complete molting takes place.
Blood masks of the swallows and Drosophila individuals were obtained in the summer of 2005. Regionswith different levels of radiation contamination wereselected for the study. Thus, flies were collected at threesites near the Chernobyl nuclear power plant with levelof radioactive contamination 5, 12.5, and 55.5 Cu/m2,
respectively, as well as in the cities of Kiev, Odessa,
Uman’, Lubny, Piryatin, and Bologna (Italy).
The swallows were trapped within the territory of
three villages in Kiev province that differed in terms ofradioactive contamination: (1) Pesky Village, Ivankov
District, at the border of the Chernobyl exclusion zone
(48 birds), with mean density of contamination withradioactive isotopes with respect to the three radioactive isotopes 137Cs, 90Sr, and 239Pu (151, 47.7, and
0.909 kBq/m2, respectively); (2) Dityatki Village,
Ivankov District, also at the border of the Chernobyl
exclusion zone (44 individuals), with mean density ofcontamination with respect to the isotopes 137Cs, 90Sr,and 239Pu (148, 40.3, and 0.557 kBq/m2, respectively);and (3) Zhovtneve Village, Borodyan District, selected
as a control, or provisionally “clean” region (21 birds),
with mean density of contamination with respect to theisotopes 137Cs, 90Sr, and 239Pu (26, 4.7, and 0.084 kBq/m2,
respectively). A total of 113 birds were selected for the
study.
Visible mutations were studied by examining fivegenerations of inbred lines obtained by means of individual crossings of 30 females from each natural population. In order to estimate the spontaneous level oflethal sex-linked mutations, males from the natural
populations were crossed with females from the line
C(1)DX and the sex ratio in the first generation compared to the control ratio according to the chi-square
method [6].
The presence of hybrid dysgenesis was determinedby means of gonadal dysgenesis assay (GD) on thebasis of the state of extracted gonads by visual evaluation of the degree of development of the gonads. Only
unilateral and bilateral. reduction of the ovaries and testes were included in the assay. A total of 50 individualsof each sex for all the populations and the laboratoryline Canton S. were analyzed. The GD percentage wascalculated according to the formula%GD = S %GD(1) + %GD(2),where %GD is the percentage of individuals with a single reduced ovary (respectively, testis) and %GD(2),the percentage of individuals with bilateral gonad
reduction.

Males aged 24 h were selected to estimate the resistance of the flies to the effect of radiation over thecourse of five generations of laboratory culturing offlies from the populations at the Chernobyl plant(55.5 Cu/m2) and from Bologna. These male individuals were subjected to gamma irradiation at a dose of
30 Gr, which it is believed induces the maximum quantity of changes in the genome with minimal mortality ofthe organisms. The radiation power amounted to
0.017 Gr/s at room temperature (Issledovatel’ gamma
radiation device, 60Co source). The irradiated males
were then crossed with females from the line C(1)DXand the sex ratio estimated in the first generation; theratio was compared with the control according to thechi-square method.
Blood was taken from the axillary vein. The masks
were dried in open air and stained for 5 min in a prepared May-Grunwald stain fixative. Final staining was
performed in a 4% aqueous solution of RomanovskiiGiemsa stain for 40 min. With such a combination it
became possible to obtain good staining of the nuclei
and differentiated staining of the granules in the granulocyte.
The frequency of erythrocytes with micronuclei(MR) and the following two nuclear anomalies which
we have described previously [7] were identified underthe microscope: “caudate” nuclei (CN) and “sprouting”
nuclei (SN). Bilaciniate nuclei (BN) and binuclear
erythrocytes (BE) were selected as indicators of disturbances in mitosis and were combined into a single
parameter in view of the strict interdependence ofchanges in these markers. Micronuclei, caudate nuclei,
sprouting nuclei, and binuclear cells are classical indicators of chromosomal instability and are therefore recommended for estimating this parameter [8]. A total of
10000 erythrocytes in each sample, which included
only mature and undamaged erythrocytes, werecounted.
Statistical analysis was performed by means of standard methods [6].
RESULTS AND DISCUSSION
Samples were collected in different regions in
Ukraine, the size of each of the samples corresponding
to the population density of D. melanogaster at thesesites (cf. table). The sample of the Italian population
was represented by flies that had been sent for the analysis and it is possible that they do not reflect the actual
parameters of this population.
In neither of the natural populations were visible
mutations discovered. On the one hand, this may berelated to the size of the samples, since according to thedata of Gershenzon [9], the frequency of visible mutations in the Kiev population in the 1937–1947 periodamounted to 0.25% and, consequently, our sample may
have been too small. On the other hand, the frequencyof visible mutations in the population of the city of
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MUTATION PROCESSES IN NATURAL POPULATIONS OF Drosophila melanogasterUman’ varied in different years, from 0.17% (1930–
1940) [9] to 0.9% (1981–1991) [10] and, consequently,
this leads us to conclude that in 2005 the frequency ofvisible mutations in this population was less than in theprevious years. It may be suggested that at the time ofthe study these populations were not in a state of mutational outbreak.
It is known that 80% of all spontaneous mutations in
D. melanogaster are caused by the activity of mobile
elements [11]. The rate of reduction of gonads in
Drosophila is one indicator of this activity. In our studies this indicator varied from 0 (which meant that we
could not consider the indicator if it was less than 1%)
to 23 ± 6%. Such a level of reduction of the gonads lead
us to conclude that in all the populations studied,including the laboratory line of the wild type Canton S.,
we had not observed any activity of the mobile elements, though it should be noted that the phenomenonof gonad reduction is itself more often found in females
in the Odessa, Chernobyl, Piryatin, and Lubny populations.
The result of an analysis of the reduction in the
gonads of both sexes provided indirect confirmation of
the low frequency of visible mutations in the populations being studied, since it had been previously demonstrated for the Uman’ population that mutation outbreaks are accompanied by the involvement of mobile
elements (P, hobo) at particular loci of the genome of
D. melanogaster.
The next stage of the study was to investigate thelevel of lethal mutations in the X-chromosome of
Drosophila. Our results show that the level of sexlinked lethal mutations exceeds statistically reliably the22control ( χ ( Lubny ) = 5.4, p  0.50; χ ( Piryatin ) = 4.02, p >0.50) only in the natural populations of Lubny andPiryatin. In the populations of Kiev, Chernobyl,
Uman’, Odessa, and Bologna, the indicator was at thelevel of the control.
Males who were descendants of individuals that had
been collected in Chernobyl and Bologna were subsequently subjected to acute irradiation with gamma rays
at a dose of 50 Gr. They were then tested for the presence of lethal mutations in the X-chromosome. It
turned out that a statistically reliable increase in thelevel of lethal mutations was observed in individuals
from the population of Bologna, whereas no suchincrease was observed among individuals from the
Chernobyl population.
In the transition to culturing of populations, anincrease in the frequency of mutations in the generations was observed, moreover, a monotonic increase inthis indicator was observed in two of the populations(Piryatin, Chernobyl, 55 Ci/m2). In the other populations this process was intermittent. Thus, besides generations in which an increase in the frequency of mutations was observed, there were also generations inwhich no mutations at all were detected. It should be
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Number of Drosophila melanogaster individuals captured
in Nature
Natural Populations

Males

Females

Kiev
Uman’
OdessaChernobyl (city), 5 Ci/m2
Chernobyl, 12.5 Ci/m2
Chernobyl, 55.5 Ci/m2
Lubny
Piryatin
Italy

208
478
621
80
34
41
226
484
139

138
704
497
70
51
35
597
723
94

emphasized that in the fifth generation the frequency ofmutations in the Chernobyl population (55.5 Ci/m2)
had grown fully ten-fold (Fig. 1).
The following mutations were found among Drosophila individuals in all the populations studied: changes
in eye color; disturbances in sclerotization of the abdomen; blisters on the wings; and changes in venation. Itshould be noted that the range of mutations in the Kiev
population was basically identical to that described by
Gershenzon et al. [9], though we did not observe certain
types of mutations in the course of five generations ofinbred crossing. Thus, we found a contraction of therange of eye color and a decrease in the diversity ofchanges in the venation of the wings. The mutations
yellow, singed, and abnormal abdomen have been previously described in several studies [10, 13–15] for the
Uman’ population, though in our experiments we did
not discover individuals with similar changes in pheno9

Kiev
Uman’
OdessaChernobyl (city), 5 Ci/m2
Chernobyl, 12.5 Ci/m2
Chernobyl, 55.5 Ci/m2
Lubny
Piryatin
Italy

8
7
6
5
4
3
2
1
0

PP

F1

F2

F3

F4

F5

Fig. 1. Frequency of mutations (along the vertical, %) infive generations of laboratory culturing: PP, parent generation; F1–F5, daughter generations.

270
‰
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0

KOZERETSKAYA et al.

Pesky
Dityatki
Zhovtneve

MN

SN

CN

BN + BE

Fig. 2. Levels of parameters of chromosomal instability
among swallows in areas with different levels of radiation
contamination: MN—micronuclei; SN—sprouting nuclei;
BN—bilaciniate nuclei; CN—caudate nuclei; and BE—
binuclear erythrocytes.

type in any of the generations in the course of culturingof the Uman’ population.
The frequency of all the parameters of chromosomal
instability studied here in the swallows were found at arather low level, which is characteristic of all the species of birds we have previously analyzed [16]. In mostof the studies micronuclei represented the basic interphase indicator of the presence of a mutagenic load
and, correspondingly, level of chromosomal instability.
The value of this indicator amounted to 0.007 ± 0.004%
in the population of swallows from an intensively contaminated region (Pesky Village), and 0.043 ± 0.020%
from a moderately contaminated region (Dityatki Village), whereas in the population of birds from a provisionally clean region (Zhovtneve Village), it reached0.090 ± 0.062%. Despite the clear decreasing trend in
the frequency of micronuclei with an increase in the
degree of contamination, the differences between the
populations with respect to this parameter were not statistically significant (Fig. 2).
Additional parameters that reflect the presence of achemical (sprouting nuclei) or physical (caudate
nuclei) mutagenic load were also estimated in three
populations of swallows. Swallows from Pesky Village
were characterized by the lowest level of these parameters (Fig. 2). The differences between these parameters were statistically reliable for both parameters (p <0.05) when compared with values obtained for birds
from moderately contaminated regions (Dityatki Village) and were comparable with values obtained in ananalysis of a population of birds from the provisionallyclean region (Zhovtneve Village). In comparing theswallows from the villages of Dityatki and Zhovtneve,a statistically significant increase in the level wasobserved only with respect to CN (0.052 ± 0.010%,
Dityatki; 0.010 ± 0.010%, Zhovtneve, p < 0.05). There
were no differences with respect to SN. A analogous
law was noted in comparing the indicators for disturbances in mitosis (BN + BE). Thus, reliably reduced

rates of this parameter were characteristic of the population of swallows from Pesky Village; swallows fromthe villages of Zhovtneve and Dityatki exhibited similarfrequencies of manifestation of disturbances in mitosis(Fig. 2). We adopted the frequencies of bilaciniate
nuclei and of binuclear erythrocytes as indicators of
disturbances in mitosis, since BN reflects amitotic divisions and BE, a disturbance in cytokinesis.
From the results that have been presented here it isclear that the erythrocytes of swallows from maximallycontaminated regions are characterized by reduced (bycomparison with the moderately contaminated zone)
frequencies of interphase manifestations of chromosomal instability and possess levels of these indicatorsthat are comparable to those of birds from the provisionally clean zone (except for BN + BE, p < 0.05).
We should note that in the moderately contaminatedzone (Dityatki) the highest level of the marker of radiation effects, that is, the number of caudate nuclei bycomparison with that in the highly contaminated zone,
was observed. It may be suggested that this phenomenon is the result of practically complete elimination ofradiosensitive individuals in the maximally contaminated regions.
Thus, our results attest to the possible existence ofan opposite dependence of the level of chromosomal
instability in swallows and that of the rate of lethal
mutations in the sex chromosome of Drosophila on thedensity of radioactive contamination. The phenomenon
has several possible explanations:
1. Rigid selection in the contaminated areas against
individuals exhibiting an increased level of chromosomal instability and an increased level of lethal mutations.
2. Adaptation of organisms living in the contaminated areas to chronic doses of radioactive effects.
3. Migration of individuals with increased levels ofchromosomal instability to cleaner regions (only forbirds).
There are preliminary data that support the second
and third explanations (Mousseau, Beckham, et al.,unpublished data). But it must also be noted that the
population of Drosophila from the region possessing
the highest radiation contamination is characterized by
the greatest mutation load. It may be suggested that theincreased heterzygosity of this population also attests
to increased adaptivity of the population.
We believe that additional studies of other regions,an analysis of several generations of birds and Drosophila, as well as studies of particular birds over thecourse of several years are needed to arrive at an unambiguous answer to these questions.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to
V. Bezrukov, V. Trotta, I. Chizhevskii, A. Peklo,
A. Zalisskii, G. Milinevskii, and V. Milenko and the
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MUTATION PROCESSES IN NATURAL POPULATIONS OF Drosophila melanogaster

research staff of the Department of Biology of Odessa
University for assistance in the collection of the subjects, as well as to N. I. Gushche for assistance in theformulation of the experiment.
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