Behavioral Ecology and Sociobiology
Stress levels of dominants reflect conflicts with subordinates in a cooperatively breeding species
Aurélie Cohasa+, Benjamin Reya, Valentine Federicoa, Corinne Regisa, Sophie Lardyb,c, Coraline Bichetd
a
UMR-CNRS 5558, Laboratoire Biométrie et Biologie Évolutive, Université Claude Bernard
Lyon 1, CNRS, F-69100, Villeurbanne, France. b
c
UMR-CNRS 6282 Biogeosciences, 21000 Dijon, France.
UMR-CNRS 5175 Centre d’Ecologie Fonctionnelle et Evolutive, Montpellier, France.
d
Institut für Vogelforschung "Vogelwarte Helgoland" (Institute of Avian Research),
Wilhelmshaven, Germany.
+
Correspondance: A. Cohas: Laboratoire Biométrie et Biologie Évolutive, UMR-CNRS 5558,
UCB Lyon 1, Bat. Grégor Mendel, 43 bd du 11 novembre 1918, 69622 Villeurbanne Cedex, France. E-mail address:
[email protected]. Phone number: +33 (0)4 72 44 85 83.
Supplementary Material S1 Social status effect on leukocyte profiles
Introduction As recommended by Davis et al. (2008) and Johnstone et al. 2012, we investigated whether the patterns of neutrophil to lymphocyte (N:L) ratio observed in the present manuscript could have been influenced by immune response. Indeed, activation of an immune response lead to profound modifications of white blood cells both quantitatively (the leukocyte concentration) and qualitatively (the leukocyte profile), and these are likely to alter the N:L ratio but without providing any direct information on stress level. Accordingly, we measured several haematological parameters attesting of the immune status but supposedly not directly influenced by stress: the leukocyte concentration, the number of eosinophils and the number of monocytes. Leukocyte concentration is a key indicator of diseases, infection and inflammation. Eosinophils play a role in the inflammation process and are mainly associated with defence against parasites (Jain 1993). Monocytes are long-lived phagocytic cells associated with defence against infections and bacteria (Roitt et al. 2001). A concomitant change in those haematological parameter and N:L ratio would indicate that immunological response, rather than stress, is the main diver of the N:L ratio patterns.
Determination of leukocyte profiles From 2009 to 2015, we drawn blood samples from the great saphenous vein of each trapped individual. We determined the leukocyte concentration using two different methods. Between 2009 and 2014, we transferred 20μl of heparinized blood in an eppendorf tube (dilution 1:20) containing a kit solution (Leuko-tic blue 1:20 plus, Bioanalytic, Germany), immediately after
collection. This solution lyses the erythrocytes and stains the leukocyte nucleus. We then carried leukocyte counts, under microscopy (x40), in a hemocytometer (Malassez cell) on a volume of 0.5μl. For the season 2015, we determined the leukocyte concentration using an automated cell counter (TC20, BioRad) following the instruction manual. We realized all leukocyte numerations within the 24 hours following blood sampling. We obtained the numbers of eosinophils and of monocytes from a drop of blood immediately smeared onto a slide and air dried. We used the May Grünwal Giemsa staining method (Duhamel and Duhamel 1984; Duhamel and Duhamel 1989) for differential counting of blood cells using an aerospray stainer (Aerospray Hematology Slide/Cytocentrifuge 7150, ELITechGroup, Wescor, France). A single observer screened all the smears by using a light microscope with 10x ocular and 100x oil immersion lenses until up to 100 leucocytes were encountered and classified as polynuclears (neutrophils, eosinophils and basophils) and mononuclears (lymphocytes and monocytes) according to (Hawkey and Dennett 1989)’s criteria.
Statistical analyses First, we tested whether the N:L ratio correlated with the leukocytes concentration and the numbers of eosinophils and monocytes, using Spearman’s rank correlation coefficients. Second, to investigate whether the leukocyte profiles differed between dominants and subordinates, we entered the leukocyte concentration (N = 178) and the numbers of eosinophils (N = 299) and monocytes (N = 299) as the dependent variables and the social status as an explanatory variable in three Generalized Linear Mixed Model (GLMM) with a logarithm link and a variance given by, respectively, a gamma, a poisson and a negative binomial distribution with an offset set to the number of leukocytes counted for the two last models. Additionally, we entered the sex, the body mass and the age of the individual as well as the year and the date of capture as potential confounding explanatory variables with all the
continuous variables being standardized. We considered additive effects of all these variables as well as the first order interactions between the year and the date of capture and between all individual variables and the social status. To control for a potential “territory” effect and pseudoreplication due to a given individual being sampled several times over years (median = 1.00, mean = 2.01, min = 1, max = 5), we entered the territory and the identity of the individual as random intercepts.
Results The N:L ratio varied neither with the leukocyte concentration (rhoSpearman = -0.05, N = 173, p = 0.55) nor with the number of eosinophils (rhoSpearman = -0.05, N = 298, p = 0.43). However, a low but significant negative correlation was found between the N:L ratio and the number of monocytes (rhoSpearman = -0.12, N = 295, p = 0.04). The social status had no effect on leukocyte concentration (β = 0.11 ±0.12, Z = 0.91, p = 0.36, Table 1), on the eosinophil (β = 0.04 ±0.15, Z = 0.24, p = 0.81, Table 1) or monocyte numbers (β = 0.08 ±0.14, Z = 0.57, p = 0.57, Table 1).
Conclusion Since N:L ratio did not strongly vary as a function the different measures of an immune response and no effect of the social status on any of the measured immune parameters was found, we can conclude that the significant effect of the social status on the N:L ratio did not result from different immune status between subordinates and dominants.
Table 1 Effect of social status on leukocyte concentration, number of eosinophils and number of monocytes. Non-significant interactions were removed from the models. Significant effects are in bold. Independent variables Fixed effects
Leukocyte concentration Estimate ± SE
Z
16.52 ± 0.28
58.28
0.11 ± 0.12
Sex (male)
Number of eosinophils Estimate ± SE
Z
