DOI: 10.5061/dryad.dbrv15f43
title: Humoral immunity of voles Cricetidae Rodentia in Siberia

description: We studied the seasonal variation of adaptive humoral immunity (AHI) in
 northern red-backed vole (Clethrionomys rutilus Pallas, 1779, RBV) and
 grey-sided vole (C. rufocanus Sundevall, 1846, GSV) in Tomsk region of
 Western Siberia. Immunoresponsiveness (IR) to sheep red blood cells was
 assessed by the number of antibody-producing cells in the spleen. The use
 of a generalized linear model to analyze the effects of species, sex, year
 of research, and season of withdrawal of individuals from nature on IR
 showed a significant effect of species identity, season of animals capture
 and the interaction of species with season. The RBV demonstrated higher
 immune responses during a year, and both species had higher IR in winter.
 Suppression of IR in spring was greater, started earlier and lasted longer
 (March–May) in GSV. In RBV, immunosuppression was restricted to April. The
 significant negative within year correlations of IR with body mass and
 masses of reproductive organs in GSV a tradeoff between AHI and growth and
 reproduction processes. A reason for the difference between species in the
 seasonal variation of AHI may be related to the difference in
 tropho-energetic requirements of each vole species. GSV is a predominantly
 herbivorous rodent and its thermoregulation seems less efficient than of
 RBV. The deeper spring immunosuppression in GSV may explain in part its
 higher mortality during the season of colds.
description: We caught voles to assess
 immunoresponsiveness from September to June in 2016–2017 and in 2017–2018
 within one habitat type (mixed coniferous – parvifoliate forest: upper
 layer: <em>Picea obovata</em> + <em>Betula
 pendula</em> + <em>Abies sibirica</em>+
 <em>Populus tremula</em>+ <em>Pinus</em>
 <em>sibirica </em>; middle layer: <em>Prunus padus
 </em>+ <em>Sorbus aucuparia sibirica</em>; undergrowth:
 <em>Ribes rubrum </em>+ <em>Ribes nigrum </em>+
 <em>Rosa majalis</em>.+ <em>Rubus idaeus</em>).
 Temperature conditions at the voles’ trapping site (<em>t
 C°</em>) averaged for every 10 days during 2016–2017 and 2017–2018
 are represented in Fig. 1. The temperatures were measured from October to
 May in the open air, in the litter and in the soil at a depth of 15 cm
 every three hours by autonomous recorders DS1921G-F5
 (Maxim Integrated
 Products, USA). Data on
 temperature were provided by S.I. Gashkov. 
 During the period of our study
 of immunoresponsiveness (2016–18), the relative abundance (N individuals
 per 100 trap-days) of the red-backed vole continued to increase in 2016,
 the growth stopped in 2017, and in 2018 there was a slight decrease in the
 number. In the gray-sided vole, a slow increase in the relative abundance
 occurred in 2016–17; the growth stopped in 2018 (Fig. 2). In the period
 2016–18, both populations demonstrated abundances close to long-term
 averages. Animals we removed from nature were kept
 individually in cages (25×40×12 cm) under the natural photoperiod at a
 temperature of +8 – +10o С from October to April, and at natural ambient
 temperatures for the remainder of the study. Food (oats, apples, grass),
 water and nesting material were provided <em>ad
 libitum</em>. We weighed the animals immediately after capture
 within an accuracy of 0.1 g and estimated the masses of the reproductive
 organs, the testes in males and the uterus with ovaries in females, with
 an accuracy of 0.001 g after killing no later than one week after capture.
 To assess the between-month variation of the masses of reproductive organs
 in order to increase the sample size, we used data for a longer period:
 for red-backed vole from 2016 to 2018 (82 males and 59 females), for
 grey-sided vole from 2016 to 2021 (93 males and 80
 females). <strong>Measurement of adaptive humoral
 immunoresponsiveness.</strong><strong> </strong>We immunized voles
 from 8 to 9 am 36 hours after capture. Tests of the duration of the glucocorticoid
 response to manipulations similar to trapping disturbance and to injection
 of ACTH in a congeneric species, <em>Clethrionomys
 glareolus</em>, showed that the effect of the stress factors ends
 within a day (Zavyalov et al., 2003; Rogovin and Naidenko, 2010). We did
 not treat pregnant females. To assess adaptive humoral immunity, we used
 local hemolysis in a liquid medium (Cunningham, 1965). We estimated the
 number of antibody-producing cells (APC) of the spleen that were formed in
 response to the introduction of a non-replicating antigen, 0.5 ml of 2 %
 of SRBC suspension injected intraperitoneally. On the 5th day, the voles
 were killed by cervical dislocation. The suspension of spleen cells was
 prepared as described by Moshkin et al. (1998). The reaction mixture
 consisted of 500 µl spleen cell suspension, 500 µl washed SRBC (4 x 109,
 erythrocytes/ml) and 500 µl lyophilized guinea pig serum (Biomed, Perm,
 Russia) resolved with 1 ml of isotonic sodium chloride solution.
 Cunningham chambers were prepared from glass microscope slides, loaded
 with 200 µl of reaction mixture (2 chambers per individuals), and
 incubated for 2 h at 37°C before hemolysis zones were counted. We
 calculated and then averaged the number of hemolysis zones
 (antibodies-producing splenocytes) for each individual. We used two
 indicators of immune activity. To eliminate within and significant between
 species differences in body masses, the total number of APCs in the spleen
 was divided by individual body mass (Novikov et al., 2010). Estimation of
 the number of APCs per unit of body mass has an advantage over the
 estimate per unit of spleen mass, since splenomegaly is often observed in
 forest voles (10,6 % in red-backed vole and 7,7 % in grey-sided vole in
 our study). An increase in the mass of the spleen in such cases is not
 associated with an increase in the number of APCs (Kravchenko,
 unpublished). To analyze correlations of immunoresponsiveness with body
 mass and with masses of reproductive organs, we used the absolute number
 of APCs. <strong>Statistical
 procedures</strong> All statistical analyses were performed using
 STATISTICA v. 10.0 (StatSoft Inc., USA). To evaluate effects of a set of
 independent variables on the magnitude of humoral immunoresponsiveness to
 SRBC (the number of antibody-producing splenocytes per unit body mass), we
 applied Generalized Linear Model (GLZ) analysis with the normal condition
 and Log link function. The intensity of humoral immune response was
 assigned as a dependent variable. Predictors included four categorical
 variables (species, sex, year of research, and season of withdrawal of
 voles with four gradations: winter, spring, summer and fall). Two-way
 interactions between predictors were also considered. The significance of
 predictors and their interactions was tested using Wald statistics and
 Likelihood ratio test type III. To choose the best model among candidate
 models we applied the Akaike Information Criterion (AIC; Akaike, 1973).
 The models were considered to have substantial support if ΔAIC (the
 difference between the AIC score of the given model and the minimal AIC
 score) was less than 2.0. For a more detailed study of within year
 variation of the immunocompetence of each of the vole species, we studied
 the monthly variation of immunoresponsiveness to SRBC. The monthly
 variation of body mass and mass of reproductive organs (testes in males,
 and uterus + ovary in females) was also considered. We used
 Kolmogorov-Smirnov test and visual examination of histograms to examine
 distributions. The Levene’s test was used to check homogeneity of
 variances. Because the assumption of normality and homogeneity of
 variances was unconfirmed for the within year variation of
 immunoresponsiveness in the body mass and mass of reproductive organs we
 used Kruskal-Wallis ANOVA with multiple (post-hoc) comparisons of average
 ranks for each pair of groups (normal z-values were computed for each
 comparison, as well as post-hoc probabilities corrected for the number of
 comparisons for a two-sided test of significance (Siegel and Castellan,
 1988). We also used
 Mann-Whitney U-test for independent pair comparisons. Spearman’s Rank
 Order Correlation Coefficient (Rs) was used to measure linkage of
 continuous non-normally distributed data. Since our analyses were based in
 part on data from a different number of years (we analyzed the
 between-month variation in the mass of the reproductive organs of voles
 over a longer time interval, than immunoresponsiveness), we indicated the
 sample sizes in the headings to the table and figures. Tests were
 two-sided, with a significance level &lt; 0.05.