Abstract: Plasma cortisol is largely bound to corticosteroid-binding globulin (CBG), which regulates its bioavailability by restricting exit from capillaries. Levels of CBG may be altered by several factors including stress and this can influence the amount of cortisol reaching cells. This study investigated the effect of social instability on plasma concentrations of CBG, total and free (not protein bound) cortisol in horses. Horses new to our research herd ('newcomers') were confined in a small yard with four dominant resident horses for 3-4 h daily for 3-4 (n = 5) or 9-14 (n = 3) days. Jugular blood was collected in the mornings from newcomers before the period of stress began ('pre-stress'), and then before each day's stress. Residents were bled before stress on the first and thirteenth day. Residents always behaved aggressively towards newcomers. By the end of the stress period, all newcomers were subordinate to residents. In newcomers (n = 8) after 3-4 days of social stress, CBG binding capacity had fallen (P = 0.0025), while free cortisol concentrations had risen (P = 0.0016) from pre-stress values. In contrast, total cortisol did not change. In residents, CBG had decreased slightly but significantly (P = 0.0162) after 12 days of stress. Residents and newcomers did not differ in pre-stress CBG binding capacity, total or free cortisol concentrations. However, by the second week of stress, CBG binding capacity was lower (P = 0.015) and free cortisol higher (P = 0.030) in newcomers (n = 3) than in residents. Total cortisol did not differ between the groups. In conclusion social stress clearly affected the adrenal axis of subordinate newcomer horses, lowering the binding capacity of CBG and raising free cortisol concentrations. However, no effect of stress could be detected when only total cortisol was measured. Therefore, to assess adrenal axis status accurately in horses, it is essential to monitor the binding capacity of CBG and free cortisol concentrations in addition to total cortisol levels.

Abstract: The costs associated with initial conflicts could be reduced if animals can assess the fighting ability of possible future opponents by watching their contest success against other individuals. We tested this hypothesis by conducting repeated dyadic dominance trials on size-matched juvenile rainbow trout,Oncorhynchus mykiss. In the first trial a dyadic contest was `observed' by a single fish separated by a transparent divider. In the second trial, the observer was paired against either the `familiar' dominant fish or an unfamiliar dominant fish from the first trial. We predicted that observers should settle conflicts with previewed opponents faster and with less aggression than those with unfamiliar fish. This prediction was supported for observers that lost against a previewed competitor, since these fish reduced their aggression more rapidly than did unfamiliar observers. Familiar observers that won, however, showed a more rapid increase in aggression compared with unfamiliar winning observers. This suggests that, regardless of whether an observer challenges the initial dominant, this `decision' is taken more rapidly in conflicts with preassessed contestants, because of the a priori information about their fighting ability. Since preassessment could save energy and allow effort to be concentrated on contests with a high payoff/probability of winning, selection may favour preview strategies when contest competition over resources is important for fitness.

Abstract: Natural selection often promotes a mix of behavioural phenotypes in a population. Adaptive variation in the propensity to take risks might explain individual differences in shyness and boldness in humans and other species. It is often implicitly assumed that shyness and boldness are general personality traits expressed across many situations. From the evolutionary standpoint, however, individual differences that are adaptive in one context (e.g. predator defence) may not be adaptive in other contexts (e.g. exploration of the physical environment or intraspecific social interactions). We measured the context specificity of shyness and boldness in a natural population of juvenile pumpkinseed sunfish,Lepomis gibbosus, by exposing the fish to a potentially threatening stimulus (a red-tipped metrestick extended towards the individual) and a nonthreatening stimulus (a novel food source). We also related these measures of shyness and boldness to behaviours observed during focal observations, both before and after the introduction of a predator (largemouth bass,Micropterus salmoides). Consistent individual differences were found within both contexts, but individual differences did not correlate across contexts. Furthermore, fish that were scored as intermediate in their response to the metrestick behaved most boldly as foragers and in response to the bass predators. These results suggest that shyness and boldness are context-specific and may not exist as a one-dimensional behavioural continuum even within a single context.

Abstract: The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of α-lactalbumin, a homologous protein of equine lysozyme. The kinetic unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by stopped-flow circular dichroism. The observed unfolding and refolding curves both agreed well with a single-exponential function. However, in the kinetic refolding reactions below 3 M guanidine hydrochloride, a burst-phase change in the circular dichroism was present, and the burst-phase intermediate in the kinetic refolding is shown to be identical with the molten globule state observed in the equilibrium unfolding. Under a strongly native condition, virtually all the molecules of equine lysozyme transform the structure from the unfolded state into the molten globule, and the subsequent refolding takes place from the molten globule state. The transition state of folding, which may exist between the molten globule and the native states, was characterized by investigating the guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding. More than 80% of the hydrophobic surface of the protein is buried in the transition state, so that it is much closer to the native state than to the molten globule in which only 36% of the surface is buried in the interior of the molecule. It is concluded that all the present results are best explained by a sequential model of protein folding, in which the molten globule state is an obligatory folding intermediate on the pathway of folding.