We observed strong and significant relationships between several neuroendocrine variables and three common measures of aggression: territorial song (in males), time responding (in females) and flyovers (in both sexes, measured in terms of flights directly over the live decoy). In support of our assumption that the short behavioural assay did not cause appreciable changes in transcription prior to sacrifice, we did not observe any significant correlations between latency to sacrifice and transcript abundance for any gene in any brain area (all |r| < 0.18, all p > 0.36).
(a) Relationships among endocrine parameters
As expected, males showed significantly higher circulating T than females (t = 7.63, d.f. = 29, p < 0.0001); so we compared T levels with measures of sensitivity separately for each sex. T levels were related to only one measure of neural sensitivity to steroids, which was the amount of AROM mRNA hypo in males only (r = −0.72, n = 16, p = 0.016; for all other correlations, p > 0.16). The sexes did not differ in behavioural measures (t < 1.30, d.f. = 30, p > 0.20), with the exception of behaviours that are specific to one sex (e.g. songs), and the sexes did not differ in any measure of transcript abundance (t < 1.23, d.f.: 26–30, p > 0.22). Thus, we considered the sexes together when correlating transcript abundance within and among brain areas, though results are qualitatively similar if the sexes are treated separately. There were no significant relationships between brain areas in AR, AROM or ORα gene expression (all |r| < 0.25, d.f.: 26–30, p > 0.17). However, the abundance of AR, AROM and ORα were positively correlated within each brain area (AR v. AROM hypo: r = 0.64, p < 0.0001, PTR: r = 0.62, p < 0.0001, VmT: r = 0.31, p = 0.081, n = 32; AR v. ORα hypo: r = 0.69, p < 0.0001, n = 29, PTR: r = 0.54, p < 0.034, n = 15, VmT: r = 0.54, p = 0.0018, n = 31; AROM v. ORα hypo: r = 0.73, p < 0.0001, n = 29, PTR: r = 0.88, p < 0.0001, n = 15, VmT: r = 0.54, p = 0.0016, n = 31).
(b) Flyovers in males and females in relation to endocrine parameters
Partial least-squares analysis pointed to the relative abundance of AR, AROM and ORα transcript in VmT as potentially important predictors of the number of flyovers directed at the intruder; neither plasma T nor any other measures of transcript abundance were identified as explaining meaningful variance in the number of flyovers (see electronic supplementary material, table S2). GLMs revealed that more aggressive individuals expressed greater AR, AROM and ORα mRNA in VmT, and there were no sex differences in these patterns of covariation (AR GLM with sex: χ2 = 9.19, d.f. = 28, p = 0.027, AR: χ2 = 7.70, p = 0.0055, sex: χ2 = 0.30, p = 0.58, sex × AR: χ2 = 0.46, p = 0.46; AROM GLM with sex: χ2 = 5.10, d.f. = 28, p = 0.16, AROM: χ2 = 3.61, p = 0.057, sex: χ2 = 0.98, p = 0.32, sex × AR: χ2 = 0.02, p = 0.88; ORα GLM with sex: χ2 = 7.11, d.f. = 27, p = 0.069, ORα: χ2 = 5.80, p = 0.016, sex: χ2 = 0.99, p = 0.31, sex × ORα: χ2 = 0.01, p = 0.93; ). Abundance of AR, AROM and OR transcript significantly predicted variance in the number of flyovers among individuals after removing the non-significant effect of sex (AR GLM: χ2 = 8.26, d.f. = 30, p = 0.004; AROM GLM: χ2 = 4.09, d.f. = 30, p = 0.043; ORα GLM: χ2 = 6.11, d.f. = 29, p = 0.0135.
Figure 1. In both sexes, individuals performing more flights directly over the live intruder showed (a) more AR, (b) more AROM and (c) more ORα mRNA in ventromedial telencephalon (VmT). These relationships did not differ between the sexes. Abundance of (more ...)
(c) Songs in males in relation to endocrine parameters
Partial least-squares analysis revealed several endocrine variables with potential relationships with the number of songs: circulating T, AR, AROM and ORα in hypo, and OR and AROM in PTR (see electronic supplementary material, table S3). GLMs demonstrated that males singing more songs expressed more AROM and ORα mRNA in PTR (AROM: χ2 = 5.09, d.f. = 14, p = 0.024, OR: χ2 = 5.06, d.f. = 13, p = 0.024), less AROM, AR and ORα in hypo (AROM: χ2 = 9.30, d.f. = 14, p = 0.0023, AR: χ2 = 4.98, d.f. = 14, p = 0.026, OR: χ2 = 5.09, d.f. = 13, p = 0.024), and have higher circulating T (χ2 = 4.75, d.f. = 13, p = 0.029, ).
Figure 2. Relationship between song and endocrine parameters: (a) plasma testosterone, (b) relative AROM mRNA in right posterior telencephalon (PTR), (c) relative ORα mRNA PTR, (d) relative AR mRNA in hypothalamus (hypo), (e) relative AROM mRNA hypo and (more ...)
(d) Time spent responding in females, in relation to endocrine parameters
Non-parametric correlations revealed no detectable relationships between the amount of time females spent responding to the intruder instead of incubating and any endocrine parameter, except for AR mRNA in hypo. Females that spent more time responding expressed marginally less AR mRNA (ρ = −0.47, n = 16, p = 0.066; ).
Females that spent more time responding aggressively to the intruder (i.e. instead of incubating) showed less AR mRNA in hypothalamus (hypo) than females that spent relatively more time incubating during the simulated territorial intrusion.