Behavioural variation at the individual level has been shown to play an important role in animal ecology and evolution. Whereas most studies have focused on subadult or adult subjects, neonates have been relatively neglected, despite studies showing that neonates can exhibit consistent inter‐individual differences during early developmental stages. Steroid hormones, including glucocorticoids (e.g. cortisol) and androgens (e.g. testosterone), play a crucial role in foetal development and maturation and could therefore drive neonate behaviour, although this relationship is poorly understood in wild animal populations. Our study addresses these knowledge gaps by investigating the association between neonate fallow deer Dama dama fawn inter‐individual variability in behavioural response to human handling and hair cortisol and testosterone levels. We found strong evidence that individual neonates display repeatable differences in the way they cope with a risky situation during their first days of life, and showed how these differences are linked to cortisol and testosterone levels accumulated in utero. We showed that, when both steroids are included in the same model, neonates with high cortisol and low testosterone levels coped in a more proactive way with human handling (higher heart rate during handling and shorter latency to leave when released) compared to those with low cortisol and high testosterone levels (lower heart rate and longer latency to leave). These results provide novel insights into the proximate mechanism leading to neonate inter‐individual variation in a wild population of large mammals. A free Plain Language Summary can be found within the Supporting Information of this article.

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Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K + (Na + )/H + antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K + in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K + or Na + ions for H + within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K + (Na + )/H + antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment. INTRODUCTIONVibrio cholerae is a Gram-negative pathogen which causes cholera, a dangerous disease that remains a public health concern (Enserink, 2010). As it transitions between the infectious state and its environmental reservoir, the bacterium encounters a dynamic range of osmotic and pH conditions. During human infection, V. cholerae produces a potent enterotoxin, cholera toxin, which promotes accumulation of Na + and Cl 2 ions in the host intestinal lumen and, in turn, causes rapid osmotic dehydration of host tissue and profuse diarrhoea. In the environment, V. cholerae is found in many coastal and estuarine waters, where it is exposed to severe periodic changes in salinity, pH and osmolarity as variable ratios of brackish and fresh water mix at different rates (Miller et al., 1984; Singleton et al., 1982a, b). Thus, both the pathogenic and environmental lifestyles of this organism require that V. cholerae can adapt to rapidly shifting osmolarities, ionic strengths and pH values. These lifestyles require that V. cholerae possess adequate molecular mechanisms to adapt to such environmental challenges.A number of V. cholerae proteins have been described that generate, maintain or use a transmembrane gradient of cations such as Na + (Häse et al., 2001). These proteins are predicted to help the bacterium survive hypo-and hyperosmolar states in addition to exploiting the Na + gradient for solute transport, pH regulation and motility. For example, the NQR complex couples Na + export to electron transport, resulting in the generation of a sodium-motive force that can then be used for various types of membrane work (Tokuda & Unemoto, 1981Zhou et al., 1999). The V. cholerae NhaA antiporter mediates Na + /H + exchange and thus regulates Na + homeostasis at pH 8.5, conditions which are not unusual for seawater in areas where...

The existence of bacterial K+/H+ antiporters preventing the over-accumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost fifty years ago. The importance of K+/H+ antiport for bacterial physiology is widely recognized but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na+/H+ antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K+. Resistance of V. cholerae to Na+ was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K+(Rb+)/H+ exchange with pH optimum at ~7.75 with an apparent Km for K+ of 1.62 mM. In the absence of K+ it exhibited Na+/H+ antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li+ for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li+ resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li+/K+ antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of structural determinants of the substrate specificity in alkali cation exchangers.

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