Signals, carotenoids and oxidative stress

BY LORENZO PEREZ-RODRIGUEZ

Carotenoids are a large family (more than 700 different types have been described) of lipophilic molecules composed of a chain of 40 carbon atoms joined with alternating single and double bonds. This characteristic chemical structure, together with the presence and location of some specific substituent groups, is responsible of their characteristic colour (yellow, orange or red) and their functions in autotrophic organisms (the only ones able to synthesize them). Carotenoids function as photosynthetic “antennae”, transferring the energy of blue and green wavelengths to chlorophyll, thereby expanding the spectrum that can be used for photosynthesis. Also, carotenoids play a significant role as photoprotectants of some key structures in vegetal cells. When chlorophyll is degraded in senescent leaves, the presence of carotenoids is revealed by the characteristic colours of autumn.

            Animals cannot synthesize carotenoids de novo, but are able to store (and, to some extent, to modify) those ingested by diet. Carotenoids are then responsible of many of the striking external colorations displayed by animals that have captured the attention of behavioural ecologists during the last two decades. Pioneer studies of John Endler in guppies Poecilia reticulata (e.g. Endler 1983) and Geoffrey E. Hill in house finches Carpodacus mexicanus (e. g. Hill 1990) suggested that carotenoid-based ornaments could act could act as honest signals of foraging capacity and overall body condition: given that  carotenoids are a limited resource for most species, only those individuals able to forage more efficiently could display more intense colouration.

Ecophysiology comes on scene

The “foraging ability” hypothesis remained as the only explanation for the honesty of carotenoid-based signals until the publication of the seminal paper of Lozano (Lozano 1994), suggesting that carotenoids could be involved in immune response and parasite resistance. Later, von Schantz et al. (1999) incorporated these ideas into a broader context, highlighting the connections between oxidative stress and immunity and paying attention to the antioxidant properties of carotenoids. This resulted in an alternative mechanism of honesty for carotenoid-based signals (the ‘‘antioxidant role’’ hypothesis) that assumes that carotenoids are relevant antioxidants in the body, which also links it to immunostimulation. Thus, individuals potentially face a trade-off between allocating available carotenoids for self-maintenance functions and ornamental coloration. Healthier individuals would require lower amounts of carotenoids for antioxidant functions, allowing instead allocation of these pigments to ornament expression, subsequently signalling their quality to conspecifics.

The importance of oxidative stress

These hypotheses addressing the antioxidant role of carotenoids are framed into the recent interest raised by oxidative stress as a relevant factor in the evolution of life history traits. But, what is oxidative stress? Aerobic metabolism involves, as a side effect, the production of highly reactive molecules called reactive oxygen and nitrogen species (ROS/RNS). ROS/RNS possess unpaired electrons, showing a strong tendency to oxidize surrounding molecules, including relevant biomolecules like lipids, proteins or DNA, thus altering their functionality (Halliwell & Gutteridge 2007). Although most of endogenously produced ROS/RNS (about 90%) are normal by-products of mitochondrial activity during aerobic metabolism, immune response and several exogenous factors do also increase ROS/RNS production. To fight ROS/RNS and protect themselves from oxidative damage, organisms rely on a relatively complex antioxidant system composed of endogenously produced compounds,

including low molecular weight antioxidants, enzymes and some other proteins without enzymatic functions, plus some food-derived antioxidants. The imbalance between ROS/RNS and antioxidant defences in favour of the former is defined as oxidative stress, and is involved in relevant processes such as ageing and several degenerative diseases (Halliwell & Gutteridge 2007) Within an ecological and evolutionary context, oxidative stress may play a key role in life-history evolution because increased oxidative damage is likely to be a significant constraint in many biological processes (Costantini 2008, Monaghan et al. 2009, Metcalfe & Alonso-Álvarez 2010).

The controversial antioxidant role of carotenoids

Over the last 15 years, the “antioxidant role” hypothesis has generated considerable interest among ecologists as a compelling and exciting (although not mutually exclusive) alternative to the ‘‘foraging ability’’ hypothesis. However, recent studies have questioned the key assumption of this ‘‘antioxidant role’’ hypothesis, claiming that carotenoids are not as powerful antioxidants as initially proposed (e.g. Hartley & Kennedy 2004, Costantini & Møller 2008). Certainly, current evidences supporting these highlighted antioxidant properties are rather scarce. Also, the simple approaches to the complex nature of the antioxidant system adopted by most studies do not allow us to assess this assumption properly. As a possible alternative, it has been suggested that carotenoids are poor antioxidants but are particularly sensitive to be altered by ROS/RNS, which may result in a spurious relationship between carotenoid pigmentation and oxidative stress that is mediated by the quality of the antioxidant system (Hartley & Kennedy 2004). Another possibility to link carotenoid functions to honest signalling is that these molecules may play a relevant role during immune response, but not necessarily because of their controversial antioxidant properties (Pérez-Rodríguez et al. 2008, Pérez-Rodríguez 2009).

Therefore, the mechanisms underlying the honesty of carotenoid-based signals are still far from consensus. To solve this question we must address the problem from new perspectives, addressing the roles of these pigments in different phases of the life cycle and under different contexts. The answers are just one step ahead, but we have to explore before finding the right way to walk along.

What is our contribution?

We have developed several lines of research in order to investigate the mechanism regulating the expression of carotenoid-based traits, a key topic to understand their role as honest signals. In collaboration with Carlos Alonso-Álvarez (IREC-CSIC), Francois Mougeot (EEZA-CSIC), Julio Blas (EBD-CSIC) and Javier Viñuela (IREC-CSIC), we have shown that the red carotenoid-based coloration of the eye ring and beak of the red-legged partridge Alectoris rufa is a honest signal of body condition and reveals the levels of intestinal parasites of individuals (Pérez-Rodríguez & Viñuela 2008, Mougeot et al. 2009). Also, the carotenoid-based colouration of the red-legged partridge is a dynamic trait whose expression is maximized during the breeding season (Pérez-Rodríguez 2008), playing a relevant role in mate assessment: females mated with male whose colouration is experimentally enhanced increase their investment in reproduction (Alonso-Álvarez et al. 2012). As found in other species with carotenoid-based traits, the colouration of the red ornaments of red-legged partridges is related to circulating levels of carotenoids (Pérez-Rodríguez & Viñuela 2008, Pérez-Rodríguez et al. 2008). But, in addition, we showed that circulating carotenoids were increased when testosterone levels were elevated (Blas et al. 2006). Nevertheless, the effects of this hormone depend on the age of the individual: in young males an elevation of testosterone levels results in increased both circulating carotenoids and redness. By contrast, the same hormonal treatment did not affect any of these variables in senescent males, that in turn suffered an immunosupressive effect caused by the hormone that was not evidenced in youngs (Alonso-Álvarez et al. 2009). We have also shown that mounting a cell-mediated immune response consumes a significant proportion of circulating carotenoids, although carotenoids do not seem to contribute much to the antioxidant barrier of plasma (Pérez-Rodríguez et al. 2008). Despite this result, the carotenoid-based coloration of the red-legged partridge seems to be linked to the antioxidant system because the pigmentation of the individual predicts its resistance to oxidative damage during such immune challenge (Pérez-Rodríguez et al.  2010).

Our research on red-legged partridges has been conducted mainly in captivity. However, we have also investigated the relationships between carotenoids, colouration and oxidative stress in the wild, using the red grouse Lagopus lagopus scoticus as model species. In collaboration with Francois Mougeot (EEZA-CSIC), Jesús Martínez-Padilla (MNCN-CSIC) y Stephen M. Redpath (Univ. of Aberdeen) among other colleagues, we have shown in a field experiment that both circulating carotenoids and the  red carotenoid-based colouration of the comb (sexual signal in the red grouse) are negatively affected by intestinal parasites (Martínez-Padilla et al. 2007), an effect mediated by oxidative stress (Mougeot et al. 2010). Testosterone enhances carotenoid allocation to combs, increasing their redness, although this effect is not always matched to a parallel increase in circulating levels of the pigment  (Mougeot et al. 2007, Martínez-Padilla et al. 2010). However, such testosterone-mediated increase in ornament redness entails a significant cost: an increase of oxidative damage (Mougeot et al. 2009).

As stated above, in order to reach a complete view of the function and evolution of carotenoid-based traits it is necessary to study the role of these pigments across tissues and phases of the life cycle. At present, using the spotless starling Sturnus unicolor as model species, we are exploring the relationship between beak colour, carotenoids, hormones and oxidative stress in adults. Also, we are studying the role of egg yolk carotenoids and their effects on embryo and nestling development. As nestlings of many other passerine species, spotless starlings exhibit conspicuous bright yellow (carotenoid-based) gapes that play important roles in begging behaviour. We are now exploring the relationships between gape development, hormone profile and oxidative stress during this period of fast development.

References

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