common vole, Microtus arvalis sensu stricto Pallas,. 1779, form obscurus (2n = 46, NFa = 68) and M. rossi- aemeridionalis Ognev, 1924 (2n = 54). Although their.
Doklady Biological Sciences, Vol. 400, 2005, pp. 48–50. Translated from Doklady Akademii Nauk, Vol. 400, No. 3, 2005, pp. 419–422. Original Russian Text Copyright © 2005 by Gileva, Polyavina, Yalkovskaya.
GENERAL BIOLOGY
Immune Hematological Characteristics and Immune Instability in Twin Species of Common Vole (the Microtus arvalis group) at Different Levels of Anthropogenic Stress E. A. Gileva, O. V. Polyavina, and L. E. Yalkovskaya Presented by Academician V.N. Bol’shakov May 12, 2004 Received May 25, 2004
The relationship between immunity and mutation process is complicated: on the one hand, mutations are necessary for the formation of cellular immunity; on the other hand, the immune system participates in sustaining genetic homeostasis by the elimination of cells carrying mutations. Therefore, it is clear that the factors changing the immune status of the body must be taken into account when studying the mutation process in natural populations. These factors include first of all infectious agents which animals constantly encounter under natural conditions, as well as mutagens occurring in the environment as a result of human activity. Therefore, we studied the degree of the damage of the chromosomal apparatus and some immunological and hematological parameters in two chromosomal twin species of common vole, Microtus arvalis sensu stricto Pallas, 1779, form obscurus (2n = 46, NFa = 68) and M. rossiaemeridionalis Ognev, 1924 (2n = 54). Although their existence as separate species has been confirmed by hybridological experiments and cytogenetic analysis, they exhibit a high degree of phenotypic similarity, inhabit the same biotopes, and are sympatrically distributed [1]. The purpose of this study was to compare the responses of their genomes and immune systems to identical stress factors in common habitats of the two species. Voles from both species were captured in the same plot about 1 ha in area in the northern outskirts of the city Ioshkar-Ola in the middle Volga region. In addition, we studied common voles from another urbanized area, a suburb of the city of Yekaterinburg, as well as M. arvalis from the Predural’e Sanctuary (Perm oblast) and from a laboratory colony originating from voles captured near a biological station of the Ural State University off Yekaterinburg, which were not subjected to noticeable anthropogenic stress. Metaphase chromosome preparations were made from bone marrow and
Institute of Plant and Animal Ecology, Ural Division, Russian Academy of Sciences, ul. Vos’mogo Marta 202, Yekaterinburg, 620144 Russia
stained by the standard method. Fifty metaphase cells per animal were analyzed. We recorded structural chromosome aberrations, numerical aberrations (aneuploidy and polyploidy), and gaps. True chromosome breaks were distinguished from gaps using generally accepted criteria (a shift relative to the chromatid axis and/or a break wider than the chromatid). Immune hematological parameters were estimated by the standard methods [2]. The radionuclide content of the bone and muscle tissues of the rodents was determined by the radiochemical and β-radiometric methods; and the concentrations of heavy metals in the liver, by means of atom absorption spectrometry. Factor analysis and the nonparametric Kruskal–Wallis (H) and Wilcoxon– Mann–Whitney (U) tests were used for statistical treatment. The differences between the groups of voles with respect to all parameters except for the frequency of aneuploid and polyploid cells were highly significant (H = 18.80–98.19, p < 0.001) (Table 1). Common voles from the Ioshkar-Ola and Yekaterinburg suburbs did not differ significantly in three cytogenetic parameters (U = 260.0–309.0, p = 0.32–0.95). However, the mean frequencies of bone marrow cells with chromosome aberrations were higher in M. arvalis from urbanized areas than in animals from the “clean” Predural’e Sanctuary (U = 68.0–176.0, p < 0.05), and the number of gaps was considerably larger in M. arvalis from Ioshkar-Ola and Yekaterinburg than in voles from the laboratory population (U = 87.0–96.0, p < 0.01). Note that increasingly much evidence is published indicating the common nature of gaps and structural chromosome aberrations, as well as the effectiveness of gaps as markers of genomic instability [3]. The increase chromosomal instability of common voles from suburbs compared to the control was accompanied by increase in the relative weight of the thymus and total number of neutrophils (with the neutrophil pool becoming more juvenile, i.e., the proportion of band neutrophils increasing) and decrease in the cellularity of the spleen and bone marrow and total number of blood leukocytes, mainly due to eosinophils, lymphocytes, and plasma cells. In addition, M. arvalis from urbanized areas, but not control
0012-4966/05/0102-0048 © 2005 Pleiades Publishing, Inc.
IMMUNE HEMATOLOGICAL CHARACTERISTICS AND IMMUNE INSTABILITY
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Table 1. Mean values of cytogenetic and immune hematological parameters in voles from the M. arvalis group Ioshkar-Ola suburb
Yekaterin- Predural’e Laboratory burg suburb Sanctuary colony
Parameter
H M. rossiaeM. arvalis meridionalis
Number of animals
28
39
M. arvalis 16
M. arvalis 14
M. arvalis 24
Percentage of cells, %: with chromosome aberrations
6.00
2.51
2.50
0.86
1.52
aneuploid and polyploid
0.71
0.36
0.38
0.14
0.34
28.68*** 7.98
with gaps
7.64
2.67
4.75
2.14
1.08
41.25***
Spleen index
3.00
3.33
5.28
2.87
0.93
64.59***
Thymus index
1.07
1.56
0.79
0.39
0.83
29.12***
the spleen
1.94
2.32
1.28
2.02
2.56
34.80***
the thymus
1.86
2.79
1.89
2.63
3.54
29.42***
the bone marrow
0.48
0.57
0.30
0.65
0.42
30.73***
3.90
3.60
3.16
5.48
5.44
27.84***
0.62
1.70
0.40
1.33
1.09
18.80**
Band neutrophils, %
16.39
18.22
40.60
10.75
14.96
31.77***
Segmented neutrophils, %
26.88
13.54
16.80
11.17
17.91
21.95**
Neutrophils, %
43.27
31.76
57.40
21.92
32.87
25.90***
Monocytes, %
1.08
1.11
0.33
0.92
2.35
98.19***
Lymphocytes, %
55.15
65.30
40.13
75.92
62.96
25.71***
Plasma cells, %
0.38
0.57
0.53
1.17
0.00
31.22***
Granular lymphocytes (azurocytes), %
1.73
1.03
1.00
0.00
0.00
39.22***
Cellularity of
Number of leukocytes,
103
in 1 µl
Eosinophils, %
** p < 0.01; *** p < 0.001.
animals, had lymphocytes with azure-positive granules (azurocytes), which are sometimes found in voles from the genus Microtus and are considered to be killer cells [4]. Chromosomal twin species inhabiting the same Ioshkar-Ola suburb significantly differed from each other in the proportions of cells with structural chromosome aberrations and with gaps (U = 270.0 and U = 171.0, respectively, p < 0.001). These parameters in M. rossiaemeridionalis were substantially higher than in M. arvalis and also considerably higher than the background values described for M. rossiaemeridionalis in the Urals (0.5–1.5%) [5]. In addition, M. rossiaemeridionalis differed from M. arvalis from the same habitat in a higher proportion of aneuploid and polyploid cells (U = 440.0, p = 0.07) and larger relative numbers of neutrophils (U = 304.5, p = 0.01) and granular lymphocytes (U = 353.0, p = 0.06), whereas the total number of lymphocytes in M. rossiaemeridionalis was decreased (U = 342.0, p = 0.05). The increased genomic instability of voles in urbanized areas is not surprising. First of all, we should take DOKLADY BIOLOGICAL SCIENCES
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into account that the environment there is contaminated with various technogenic mutagens. Therefore, we compared the voles from suburbs and the sanctuary with respect to the copper, zinc, cadmium, and lead contents of the liver, as well as the concentrations of 40K, 90Sr, 137Cs, 226Ra, and 232Th in the bone and muscle tissues. We did not find any differences between species or populations in these parameters; their values were within the limits of the regional normal range described for the Urals [5]. Apparently, the high genomic instability of voles from Ioshkar-Ola and Yekaterinburg is strongly related to the effect of organic pollutants that inevitably contaminate the environment of big cities and are known as potent clastogenic agents. The relationship of the chromosome aberration rate with immune hematological parameters and some population demographic characteristics was studied with the use of factor analysis, which allowed us to find the factors that accounted for 57% of the observed variance (Table 2). Cytogenetic parameters, except for aneuploidy and polyploidy, make the main contribution to the first factor and a grouped together with the factors of
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GILEVA et al.
Table 2. The results of the factor analysis of cytogenetic and immune hematological parameters in voles from the M. arvalis group Parameter
Factor 1
Factor 2
Factor 3
Factor 4
Locality Species Foot index (an indicator of age) Participation in reproduction Spleen index Thymus index Leukocytes Eosinophils Neutrophils Monocytes Lymphocytes Plasma cells Azurocytes Chromosome aberrations Aneuploidy and polyploidy Gaps
0.714 0.684 0.436 0.189 0.293 0.145 –0.306 –0.146 0.545 –0.261 –0.510 0.106 0.596 0.703 0.233 0.682
–0.312 –0.077 –0.763 0.679 0.341 –0.842 0.463 0.005 0.536 0.252 –0.546 0.056 0.240 –0.109 0.200 0.031
0.160 0.101 –0.049 0.522 0.310 –0.171 –0.027 0.474 –0.579 –0.184 0.571 0.197 0.203 0.005 0.138 0.165
–0.107 0.308 –0.071 –0.185 –0.444 –0.123 0.113 –0.256 –0.204 0.535 0.213 0.377 0.252 0.247 0.482 –0.059
Eigenvalues Proportion of explained variance
3.431 21.4
2.948 18.4
1.489 9.3
1.311 8.2
species and population, as well as the relative numbers of neutrophils, lymphocytes, and azurocytes. In other words, the increase in the frequency of chromosome aberrations is accompanied by an increased strain of the phagocytosis system, the suppression of the lymphocytic system, and the activation of killer cells (azurocytes). The shifts in the immune system are most likely to be related to the presence of infectious agents (probably persistent ones). The effect of these agents can increase chromosomal instability, because pathogens, mainly viruses, are known to induce chromosome mutations [6]. The considerably higher rate of chromosome aberrations in M. rossiaemeridionalis compared to M. arvalis from the same habitat is noteworthy. We observed a similar situation in other localities; usually, the chromosomal instability of M. rossiaemeridionalis was somewhat higher both in areas with an increased background mutagenic pollution of the environment and in zones of radiation accidents. In addition, in radiationaffected zones, M. rossiaemeridionalis exhibited more drastic disturbances in ontogenetic homeostasis expressed as the asymmetry of bilateral craniometric parameters [7]. Thus, M. rossiaemeridionalis are more sensitive to various stresses at both the genomic and the ontogenetic levels, which is one more evidence for the considerable evolutionary divergence of chromosomal twin species from the M. arvalis group.
ACKNOWLEDGMENTS We are grateful to M.I. Cheprakov, S.B. Rakitin, and I.A. Pashnina for their help in treating the material. This study was supported by the Russian Foundation for Basic Research (project no. 02-04-49071) and the Ministry of Science and Technology of the Russian Federation (project no. NSh-237.2003.4). REFERENCES 1. Malygin, V.M., Sistematika obyknovennykh polevok (Systematics of Common Vole), Moscow: Nauka, 1983. 2. Laboratornye metody issledovaniya v klinike: Spravochnik (Laboratory Methods in Clinic: A Reference Book), Men’shikov, V.V. et al., Eds., Moscow: Meditsina, 1987. 3. Paz-y-Mino, C., Davalos, M.V., Sanchez, M.E., et al., Mutat. Res., 2002, vol. 516, no. 1/2, pp. 57–61. 4. Mihok, S., Descoteaux, J.-P., Lawton, T.B., et al., Can. J. Zool., 1987, vol. 65, no. 1, pp. 54–62. 5. Gileva, E.A., Ekologo-geneticheskii monitoring s pomoshch’yu gryzunov (ural’skii opyt) (Ecological and Genetic Monitoring Using Rodents: The Urals Experience), Yekaterinburg: Izd. Ural. Inst., 1997. 6. Gileva, E.A., Polyavina, O.V., Apekina, N.S., et al., Genetika (Moscow), 2001, vol. 37, no. 4, pp. 504–510. 7. Gileva, E.A. and Nokhrin, D.Yu., Zh. Obshch. Biol., 2001, vol. 62, no. 3, pp. 217–225.
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