Detailed husbandry, field methods and molecular protocols have been published elsewhere (Madsen et al. 2000
; Olsson et al. 2000
) and we, therefore, only give a brief account of methods used. Males were captured by noose or by hand within a week of emergence from hibernation (ca
first week in May), were blood sampled within 30
s of capturing, measured and weighed and, on the day following capture, anaesthetized and given a silastic implant (every second male received an empty control implant or one with a 4
mm crystalline testosterone column; Sigma product no. T 1500; Sigma Aldrich Pty Ltd, Castle Hill, NSW, Australia). They were then individually marked with a numbered cloth adhesive sticker on their backs, released within 24
h at the place of capture, and then monitored with respect to mating success throughout the approximately 3 week mating season every day when weather permitted lizard activity. Male mating success was scored as successful mate guarding of females, which we have shown elsewhere is strongly correlated with male probability of paternity, using mini- and microsatellites (Olsson et al. 2000
). Since this measure of mating success was correlated with a male's total number of observations (rs
=40), we divided the number of observations of mate guarding by the total number of observations and used this ratio as an index of male mating success. At the end of the mating season, the lizards were recaptured and blood sampled again to assess the effect of the testosterone treatment on the target traits.
All observations in the current paper were made in 1999, with the exception of a tick removal experiment in 2002. Unpublished data show that tick resistance is a heritable trait in sand lizards (at least in females; Olsson et al.
in preparation). Therefore, in order to assess whether individual differences in tick load were repeatable in males, and potentially heritable, ticks were manually removed the first week of spring activity and were then recounted after the mating season to assess repeatability of tick counts. Although this cannot rule out differences in tick encounter rate among discrete habitats, our previous work shows that males are highly mobile through habitat types, and that mobility (metres moved during the mating season) is uncorrelated with male tick load (Olsson et al. 2000
). Thus, our removal experiment should be highly indicative of among-individual differences in tick resistance, which we know is partly genetically determined, suggesting that tick resistance is heritable and should evolve in response to selection.
MHC class I genotype was screened using a species-specific probe for RFLP (Madsen et al. 2000
). On average, males had 11.6±3.3, s.e., RFLP-fragments in their genotype. To estimate prevalence of haemoparasites, a 30
μl blood sample was spread on a microscope slide in a standardized way, air dried, fixed with methanol and Giemsa stained, and stored dry until cell counts were performed. Erythrocytes and leucocytes were counted in 50 standardized areas along four longitudinal transects on each microscope slide at 100×. In total, 226
574 leucocytes and 812
368 erythrocytes were counted and screened for haemoprotid parasites. Haemoprotid parasite counts were made by parasitologist Dr Dan Christensson, Department of Parasitology, the Swedish National Veterinary Institute (SVA), Uppsala, Sweden.
The statistical analyses were based on parametric techniques when the assumptions of normality were met. We consistently tested the directional prediction that O-males suffered higher costs in terms of altered blood cell counts, internal parasite load, and body condition than do NO-males subsequent to an initial tick load accumulated during the first week of spring activities, i.e. when males novel to haemoprotid parasites first become infected and/or already infected males may receive boost infections via tick vectors (Rheichenbach-Klinke & Elkan 1965
). The rational for this is the directional prediction that O-males, with an MHC fragment associated with tick resistance, mount a more successful, but energetically costly, immune response than NO-males. Therefore, presented p
values for the three t
-tests assessing haemocytology and parasitaemia are one-tailed.