Seasonal but not sex-biased gene expression of the carotenoid ketolase, CYP2J19, in the sexually dichromatic southern red bishop (Euplectes orix).

Intense red colours in birds are often owing to ketocarotenoids (KCs). In many land birds, KCs are oxidized from dietary yellow precursors, presumably by the avian carotenoid ketolase CYP2J19, the regulation and constraints of which have important implications for condition-dependence and honest signalling of carotenoid colour displays. We investigated hepatic CYP2J19 gene expression in the seasonally and sexually dichromatic southern red bishop (Euplectes orix) in relation to season, sex, progression of the prenuptial moult, testis size, body condition, redness and circulating sex steroids. A coloration function of CYP2J19 is supported by a seasonal upregulation prior to and during the carotenoid-depositing stage of the male prenuptial moult. However, CYP2J19 expression was similarly high in females (which do not moult prenuptially), and remained high in males after moult, suggesting additional or alternative roles of hepatic CYP2J19 or its products, such as detoxification or antioxidant functions. In males, the CYP2J19 upregulation preceded and was unrelated to the rise in plasma testosterone, but was correlated with androstenedione, probably of adrenal origin and compatible with luteinizing hormone-induced and (in females) oestrogen-suppressed moult. Finally, contrary to ideas that carotenoid ketolation rate mediates honest signalling of male quality, CYP2J19 expression was not related to plumage redness or male body condition.

Increases in glucocorticoids are sufficient but not necessary to increase cooperative burrowing in Damaraland mole-rats.

Despite widespread interest in the evolution of cooperative behaviour, the physiological mechanisms shaping their expression remain elusive. We tested the hypothesis that glucocorticoid (GC) hormones affect cooperative behaviour using captive Damaraland mole-rats (Fukomys damarensis), a cooperatively breeding mammal. Within groups, individuals routinely contribute to public goods that include foraging tunnels, which provide all group members access to the tubers of desert plants they feed on, communal food stores and nests. We found that experimental increases in glucocorticoid concentration (GCc) in non-breeding female helpers led them to be active for longer and to burrow more while active, raising their daily contributions to burrowing, but not food carrying or nest building. However, experimentally induced increases in burrowing did not lead to elevated GCc in helpers of both sexes. These results suggest that heightened GCc may stimulate some cooperative behaviours that are energetically demanding (a characteristic shared by many types of cooperative activities across species) but that the cooperative behaviours affected by GCc can also be regulated by other mechanisms.

Oxidative costs of cooperation in cooperatively breeding Damaraland mole-rats.

Within cooperatively breeding societies, individuals adjust cooperative contributions to maximize indirect fitness and minimize direct fitness costs. Yet, little is known about the physiological costs of cooperation, which may be detrimental to direct fitness. Oxidative stress, the imbalance between reactive oxygen species (by-products of energy production) and antioxidant protection, may represent such a cost when cooperative behaviours are energetically demanding. Oxidative stress can lead to the accumulation of cellular damage, compromising survival and reproduction, thus mediating the trade-off between these competing life-history traits. Here, we experimentally increased energetically demanding cooperative contributions in captive Damaraland mole-rats (Fukomys damarensis). We quantified oxidative stress-related effects of increased cooperation on somatic and germline tissues, and the trade-off between them. Increased cooperative contributions induced oxidative stress in females and males, without increasing somatic damage. Males accumulated oxidative damage in their germline despite an increase in antioxidant defences. Finally, oxidative damage accumulation became biased towards the germline, while antioxidant protection remained biased towards the soma, suggesting that males favour the maintenance of somatic tissues (i.e. survival over reproduction). Our results show that heightened cooperative contributions can ultimately affect direct fitness through oxidative stress costs, which may represent a key selective pressure for the evolution of cooperation.

No task specialization among helpers in Damaraland mole-rats.

The specialization of individuals in specific behavioural tasks is often attributed either to irreversible differences in development, which generate functionally divergent cooperative phenotypes, or to age-related changes in the relative frequency with which individuals perform different cooperative activities; both of which are common in many insect caste systems. However, contrasts in cooperative behaviour can take other forms and, to date, few studies of cooperative behaviour in vertebrates have explored the effects of age, adult phenotype and early development on individual differences in cooperative behaviour in sufficient detail to discriminate between these alternatives. Here, we used multinomial models to quantify the extent of behavioural specialization within nonreproductive Damaraland mole-rats, Fukomys damarensis, at different ages. We showed that, although there were large differences between individuals in their contribution to cooperative activities, there was no evidence of individual specialization in cooperative activities that resembled the differences found in insect societies with distinct castes where individual contributions to different activities are negatively related to each other. Instead, individual differences in helping behaviour appeared to be the result of age-related changes in the extent to which individuals committed to all forms of helping. A similar pattern is observed in cooperatively breeding meerkats, Suricata suricatta, and there is no unequivocal evidence of caste differentiation in any cooperative vertebrate. The multinomial models we employed offer a powerful heuristic tool to explore task specialization and developmental divergence across social taxa and provide an analytical approach that may be useful in exploring the distribution of different forms of helping behaviour in other cooperative species.

Sensitive and selective quantification of free and total malondialdehyde in plasma using UHPLC-HRMS.

Quantification of malondialdehyde (MDA) as a marker of lipid peroxidation is relevant for many research fields. We describe a new sensitive and selective method to measure free and total plasmatic MDA using derivatization with 2,4-dinitrophenylhydrazine (DNPH) and ultra-HPLC-high-resolution MS. Free and total MDA were extracted from minute sample amounts (10 µl) using acidic precipitation and alkaline hydrolysis followed by acidic precipitation, respectively. Derivatization was completed within 10 min at room temperature, and the excess DNPH discarded by liquid-liquid extraction. Quantification was achieved by internal standardization using dideuterated MDA as internal standard. The method's lowest limit of quantification was 100 nM and linearity spanned greater than three orders of magnitude. Intra- and inter-day precisions for total MDA were 2.9% and 3.0%, respectively, and those for free MDA were 12.8% and 24.9%, respectively. Accuracy was 101% and 107% at low and high concentrations, respectively. In human plasma, free MDA levels were 120 nM (SD 36.26) and total MDA levels were 6.7 µM (SD 0.46). In addition, we show the applicability of this method to measure MDA plasma levels from a variety of animal species, making it invaluable to scientists in various fields.

Differences in cooperative behavior among Damaraland mole rats are consequences of an age-related polyethism.

In many cooperative breeders, the contributions of helpers to cooperative activities change with age, resulting in age-related polyethisms. In contrast, some studies of social mole rats (including naked mole rats, Heterocephalus glaber, and Damaraland mole rats, Fukomys damarensis) suggest that individual differences in cooperative behavior are the result of divergent developmental pathways, leading to discrete and permanent functional categories of helpers that resemble the caste systems found in eusocial insects. Here we show that, in Damaraland mole rats, individual contributions to cooperative behavior increase with age and are higher in fast-growing individuals. Individual contributions to different cooperative tasks are intercorrelated and repeatability of cooperative behavior is similar to that found in other cooperatively breeding vertebrates. Our data provide no evidence that nonreproductive individuals show divergent developmental pathways or specialize in particular tasks. Instead of representing a caste system, variation in the behavior of nonreproductive individuals in Damaraland mole rats closely resembles that found in other cooperatively breeding mammals and appears to be a consequence of age-related polyethism.

Arginine vasotocin neuronal phenotype and interspecific cooperative behaviour.

The nonapeptide arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin are well known for their role in the modulation of several intraspecific social behaviours, such as social approach/withdrawal and aggression. Recently, we suggested that AVT might also be important in the regulation of interspecific social behaviours as it modulates interspecific cooperative behaviours in the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus. AVT injections decreased cleaners' propensity to engage in cleaning interactions with their clients, suggesting that high levels of this peptide compromise the cleaners' cooperative motivation. Therefore, we hypothesise that low endogenous levels of AVT are a prerequisite for the expression of interspecific cleaning behaviour in cleaner wrasses, since it allows them to approach and interact with interspecific individuals, and that this should be reflected in their AVT neuronal phenotype. Here we test this hypothesis by comparing the AVT neuronal phenotypes of two phylogenetically closely related species that live in similar environments but diverge in the expression of interspecific cooperative behaviour: an obligate cleaner wrasse (L. dimidiatus) and a non-cleaner corallivore wrasse (Labrichthys unilineatus). The two species are predicted to differ in their AVT neuronal phenotypes as a reflection of their ability/inability to approach and interact with interspecific individuals, with cleaners presenting smaller and/or less numerous AVT-immunoreactive (ir) neurons. A sex difference in AVT neuronal phenotypes was also predicted because males of both species appear to be more aggressive than females. As described for most of the other teleost species, AVT-ir neurons were restricted to the preoptic area, and in agreement with our first prediction cleaners presented smaller and less numerous AVT-ir neurons in the gigantocellular preoptic area (gPOA) compared to non-cleaners. Contrary to our second prediction, AVT neuronal phenotypes did not differ between sexes in either species, but differences in other features of the AVT system cannot be ruled out. In summary, the results presented here suggest a putative role for AVT gPOA neurons in the ability of a cleaner wrasse to approach and interact with a client, through their projections to extrahypothalamic brain areas.

Arginine vasotocin regulation of interspecific cooperative behaviour in a cleaner fish.

In an interspecific cooperative context, individuals must be prepared to tolerate close interactive proximity to other species but also need to be able to respond to relevant social stimuli in the most appropriate manner. The neuropeptides vasopressin and oxytocin and their non-mammalian homologues have been implicated in the evolution of sociality and in the regulation of social behaviour across vertebrates. However, little is known about the underlying physiological mechanisms of interspecific cooperative interactions. In interspecific cleaning mutualisms, interactions functionally resemble most intraspecific social interactions. Here we provide the first empirical evidence that arginine vasotocin (AVT), a non-mammalian homologue of arginine vasopressin (AVP), plays a critical role as moderator of interspecific behaviour in the best studied and ubiquitous marine cleaning mutualism involving the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus. Exogenous administration of AVT caused a substantial decrease of most interspecific cleaning activities, without similarly affecting the expression of conspecific directed behaviour, which suggests a differential effect of AVT on cleaning behaviour and not a general effect on social behaviour. Furthermore, the AVP-V1a receptor antagonist (manning compound) induced a higher likelihood for cleaners to engage in cleaning interactions and also to increase their levels of dishonesty towards clients. The present findings extend the knowledge of neuropeptide effects on social interactions beyond the study of their influence on conspecific social behaviour. Our evidence demonstrates that AVT pathways might play a pivotal role in the regulation of interspecific cooperative behaviour and conspecific social behaviour among stabilized pairs of cleaner fish. Moreover, our results suggest that the role of AVT as a neurochemical regulator of social behaviour may have been co-opted in the evolution of cooperative behaviour in an interspecific context, a hypothesis that is amenable to further testing on the potential direct central mechanism involved.