Oxidative status: A general but overlooked indicator of welfare across animal species?

Because of their ubiquity, plasticity, and direct effects on the nervous system, markers of oxidative status may be of great value to assess animal welfare across species and conditions in the wild. However, welfare biologists have not yet seized this opportunity, possibly because the validity of these markers as welfare indicators remains questionable. A validation process was, therefore, performed here using a meta-analytical approach considering three conditions assumed to impair the welfare of animals. With very few exceptions, two of the four considered markers consistently varied across these negatively-valenced conditions. By highlighting the current underrepresentation of markers of oxidative status in animal welfare studies, and by concretely illustrating that some of these markers can consistently reflect negative affective states, this article aims to encourage biologists to include these physiological markers in their toolbox to better measure, monitor, and perhaps also improve the welfare of animals in their natural habitat.

Irreversible impact of early thermal conditions: an integrative study of developmental plasticity linked to mobility in a butterfly species.

Within populations, phenotypic plasticity may allow adaptive phenotypic variation in response to selection generated by environmental heterogeneity. For instance, in multivoltine species, seasonal changes between and within generations may trigger morphological and physiological variation enhancing fitness under different environmental conditions. These seasonal changes may irreversibly affect adult phenotypes when experienced during development. Yet, the irreversible effects of developmental plasticity on adult morphology have rarely been linked to life-history traits even though they may affect different fitness components such as reproduction, mobility and self-maintenance. To address this issue, we raised larvae of Pieris napi butterflies under warm or cool conditions to subsequently compare adult performance in terms of reproduction performance (as assessed through fecundity), displacement capacity (as assessed through flight propensity and endurance) and self-maintenance (as assessed through the measurement of oxidative markers). As expected in ectotherms, individuals developed faster under warm conditions and were smaller than individuals developing under cool conditions. They also had more slender wings and showed a higher wing surface ratio. These morphological differences were associated with changes in the reproductive and flight performance of adults, as individuals developing under warm conditions laid fewer eggs and flew larger distances. Accordingly, the examination of their oxidative status suggested that individuals developing under warm conditions invested more strongly into self-maintenance than individuals developing under cool conditions (possibly at the expense of reproduction). Overall, our results indicate that developmental conditions have long-term consequences on several adult traits in butterflies. This plasticity probably acts on life-history strategies for each generation to keep pace with seasonal variations and may facilitate acclimation processes in the context of climate change.

Species-specific effects of thermal stress on the expression of genetic variation across a diverse group of plant and animal taxa under experimental conditions.

Assessing the genetic adaptive potential of populations and species is essential for better understanding evolutionary processes. However, the expression of genetic variation may depend on environmental conditions, which may speed up or slow down evolutionary responses. Thus, the same selection pressure may lead to different responses. Against this background, we here investigate the effects of thermal stress on genetic variation, mainly under controlled laboratory conditions. We estimated additive genetic variance (VA), narrow-sense heritability (h2) and the coefficient of genetic variation (CVA) under both benign control and stressful thermal conditions. We included six species spanning a diverse range of plant and animal taxa, and a total of 25 morphological and life-history traits. Our results show that (1) thermal stress reduced fitness components, (2) the majority of traits showed significant genetic variation and that (3) thermal stress affected the expression of genetic variation (VA, h2 or CVA) in only one-third of the cases (25 of 75 analyses, mostly in one clonal species). Moreover, the effects were highly species-specific, with genetic variation increasing in 11 and decreasing in 14 cases under stress. Our results hence indicate that thermal stress does not generally affect the expression of genetic variation under laboratory conditions but, nevertheless, increases or decreases genetic variation in specific cases. Consequently, predicting the rate of genetic adaptation might not be generally complicated by environmental variation, but requires a careful case-by-case consideration.

Birds of different morphs use slightly different strategies to achieve similar reproductive performance following heatwave exposure.

Responses to extreme climatic events may differ between individuals of distinct morphs which differ in life-history strategies, resulting in climate change 'winners' and 'losers' within species. We examined the reproductive performance and carry-over effects on offspring of black- and red-headed Gouldian finches Erythrura gouldiae after exposure to simulated heatwaves of moderate or severe intensity. We expected black-headed pairs' reproductive performance to decline after the severe heatwave because only the condition of black-headed females deteriorates during such a heatwave. Supporting the fact that Gouldian finches of different morphs use alternative reproductive strategies, we found that black-headed females initiated egg-laying a month earlier than red-headed females after experiencing a severe heatwave. We also found that this severe heatwave resulted in shorter spermatozoa in males irrespective of their morph. Despite these effects associated with heatwave intensity, the overall reproductive performance of both morphs was not affected by this factor, which was possibly due to an increased nestling provisioning rate by parents after exposure to the severe heatwave. However, offspring still bore the cost of parental exposure to the severe heatwave, as they showed a reduced condition (lower plasma antioxidant capacity and transient lower breathing rate) and higher oxidative damage (at least in fledglings with black-headed parents). These results suggest that inter-morph phenotypic variability in the Gouldian finch does not result in clear differences in reproductive performance following heatwave exposure, despite basal phenotypic differences between morphs. Whether animals using alternative reproductive strategies are, in the end, differently affected by climate changes will likely depend on the capacity of their offspring to recover from altered developmental conditions.

Genotype-environment interactions rule the response of a widespread butterfly to temperature variation.

Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. This issue is of special interest in the current era of rapidly changing climatic conditions. Here, we investigate clinal variation and plastic responses in life history, morphology and physiology in the butterfly Pieris napi along a pan-European gradient by exposing butterflies raised in captivity to different temperatures. We found clinal variation in body size, growth rates and concomitant development time, wing aspect ratio, wing melanization and heat tolerance. Individuals from warmer environments were more heat-tolerant and had less melanised wings and a shorter development, but still they were larger than individuals from cooler environments. These findings suggest selection for rapid growth in the warmth and for wing melanization in the cold, and thus fine-tuned genetic adaptation to local climates. Irrespective of the origin of butterflies, the effects of higher developmental temperature were largely as expected, speeding up development; reducing body size, potential metabolic activity and wing melanization; while increasing heat tolerance. At least in part, these patterns likely reflect adaptive phenotypic plasticity. In summary, our study revealed pronounced plastic and genetic responses, which may indicate high adaptive capacities in our study organism. Whether this may help such species, though, to deal with current climate change needs further investigation, as clinal patterns have typically evolved over long periods.

Differential oxidative costs of locomotory and genital damage in an orb-weaving spider.

In animals that regularly experience tissue loss, physiological responses may have evolved to overcome the related costs. Changes in oxidative status may reflect such self-maintenance mechanisms. Here, we investigated how markers of oxidative status vary in female orb-weaving spiders (Larinia jeskovi) by mimicking two distinct types of tissue loss they may naturally encounter: damage to their locomotory system and damage to their external genital structure (scapus), as inflicted by males during copulation (external female genital mutilation). Damage to the locomotory system resulted in a significant shift in oxidative status, reflecting investment in self-maintenance. In contrast, the loss of the scapus did not result in quantitative changes of oxidative markers. This lack of a physiological response suggests negligible physiological costs of genital mutilation for female spiders. However, not being able to remate with other males might be costly for females.

Clinal variation in investment into reproduction versus maintenance suggests a 'pace-of-life' syndrome in a widespread butterfly.

Extreme weather events such as heat waves are predicted to increase in the course of anthropogenic climate change. Widespread species are exposed to a variety of environmental conditions throughout their distribution range, often resulting in local adaptation. Consequently, populations from different regions may vary in their capacity to deal with challenging conditions such as thermal stress. In this study, we investigated clinal variation in body size, fecundity, and oxidative markers along a pan-European latitudinal gradient in the green-veined white butterfly Pieris napi, and additionally gene expression in German individuals. We exposed butterflies from replicated Italian, German, and Swedish populations to cold, control, or hot temperatures for 24 h. Under hot conditions, molecular chaperones were up-regulated, while oxidative damage remained unaffected and levels of the antioxidant glutathione (GSH) were reduced under cold and hot conditions. Thus, the short-term exposure to heat stress did not substantially affect oxidative balance. Moreover, we found decreased body size and fecundity in cooler compared with warmer regions. Interestingly, oxidative damage was lowest in Swedish animals exhibiting (1) high levels of GSH, (2) low early fecundity, and (3) low larval growth rates. These results suggest that Swedish butterflies have a slower life style and invest more strongly into maintenance, while those from warmer regions show the opposite pattern, which may reflect a 'pace-of-life' syndrome.

Morph- and sex-specific effects of challenging conditions on maintenance parameters in the Gouldian finch.

Intraspecific discrete polymorphism is associated with the use of alternative life-history strategies, reflected by distinct reproductive or copying strategies in individuals of different morphs. Yet, morph-specific costs and benefits related to different life-history strategies remain unclear. Here, we examined in the polymorphic Gouldian finch (Erythrura gouldiae) whether markers of somatic maintenance (body mass, oxidative status and telomere length) differed between red- and black-headed birds under energetically demanding conditions (during heatwaves of different intensity, and during moult or breeding following heatwaves). During heatwaves, red-headed birds showed a homogeneous response, as males and females initially tended to gain mass and had reduced plasma hydroperoxide levels (a marker of oxidative damage) irrespective of heatwave intensity. In contrast, black-headed birds showed a stronger and more heterogeneous response, as black-headed males gained mass at the beginning of the thermoneutral heatwave and showed stable oxidative status, whereas black-headed females lost mass and tended to show higher hydroperoxide levels at the end of the thermocritical heatwave. Following heatwaves, we found morph-specific oxidative costs owing to moult or reproduction, with oxidative markers varying only in black-headed birds. Again, oxidative markers varied differently in black-headed males and females, as plasma antioxidant capacity decreased in moulting or breeding females, whereas males showed higher hydroperoxide levels with larger broods. For the first time, our study highlights that within polymorphic species, some individuals appear more vulnerable than others when coping with energetically demanding conditions. In the context of climate change, such differential effects may ultimately alter the currently observed balance between morphs and sexes within natural populations.

Reproductive performance and diving behaviour share a common sea-ice concentration optimum in Adélie penguins (Pygoscelis adeliae).

The Southern Ocean is currently experiencing major environmental changes, including in sea-ice cover. Such changes strongly influence ecosystem structure and functioning and affect the survival and reproduction of predators such as seabirds. These effects are likely mediated by reduced availability of food resources. As such, seabirds are reliable eco-indicators of environmental conditions in the Antarctic region. Here, based on 9 years of sea-ice data, we found that the breeding success of Adélie penguins (Pygoscelis adeliae) reaches a peak at intermediate sea-ice cover (ca. 20%). We further examined the effects of sea-ice conditions on the foraging activity of penguins, measured at multiple scales from individual dives to foraging trips. Analysis of temporal organisation of dives, including fractal and bout analyses, revealed an increasingly consistent behaviour during years with extensive sea-ice cover. The relationship between several dive parameters and sea-ice cover in the foraging area appears to be quadratic. In years of low and high sea-ice cover, individuals adjusted their diving effort by generally diving deeper, more frequently and by resting at the surface between dives for shorter periods of time than in years with intermediate sea-ice cover. Our study therefore suggests that sea-ice cover is likely to affect the reproductive performance of Adélie penguins through its effects on foraging behaviour, as breeding success and most diving parameters share a common optimum. Some years, however, deviated from this general trend, suggesting that other factors (e.g. precipitation during the breeding season) might sometimes become preponderant over the sea-ice effects on breeding and foraging performance. Our study highlights the value of monitoring fitness parameters and individual behaviour concomitantly over the long-term to better characterize optimal environmental conditions and potential resilience of wildlife. Such an approach is crucial if we want to anticipate the effects of environmental change on Antarctic penguin populations.

Mind the cell: Seasonal variation in telomere length mirrors changes in leucocyte profile.

Leucocytes are typically considered as a whole in studies examining telomere dynamics in mammals. Such an approach may be precarious, as leucocytes represent the only nucleated blood cells in mammals, their composition varies temporally, and telomere length differs between leucocyte types. To highlight this limitation, we examined here whether seasonal variation in leucocyte composition was related to variation in telomere length in free-ranging mandrills (Mandrilllus sphinx). We found that the leucocyte profile of mandrills varied seasonally, with lower lymphocyte proportion being observed during the long dry season presumably because of the combined effects of high nematode infection and stress at that time of the year. Interestingly, this low lymphocyte proportion during the long dry season was associated with shorter telomeres. Accordingly, based on longitudinal data, we found that seasonal changes in lymphocyte proportion were reflected by corresponding seasonal variation in telomere length. Overall, these results suggest that variation in lymphocyte proportion in blood can significantly affect telomere measurements in mammals. However, lymphocyte proportion did not entirely explain variation in telomere length. For instance, a lower lymphocyte proportion with age could not fully explain shorter telomeres in older individuals. Overall, our results show that telomere length and leucocyte profile are strongly although imperfectly intertwined, which may obscure the relationship between telomere dynamics and ageing processes in mammals.

Socially-induced variation in physiological mediators of parental care in a colonial bird.

Social facilitation of reproduction occurs in humans and animals, and may represent one of the bases of reproduction in groups. However, its underlying physiological mechanisms remain largely unexplored. Here, we found in a colonial bird, the Adélie penguin (Pygoscelis adeliae), that the number of parental interactions (nest relief ceremonies) performed by breeding individuals on the colony was positively related to prolactin levels in other breeding individuals exposed to these interactions (i.e. focal individuals). As prolactin is typically involved in the expression of parental behaviour in birds, this suggests that parental interactions by conspecifics represent social cues that might increase parental motivation in focal individuals. Moreover, parental interactions were not related to corticosterone levels in focal individuals, suggesting that these social cues were not stressful for penguins. However, social stimulation still had a cost for focal individuals, as it was negatively related to their antioxidant defences (a component of self-maintenance). As social stimulation was also positively related to prolactin levels, this highlights the fact that social stimulation acts on the trade-off between reproduction and self-maintenance. For the first time, the results of the current study shed light on the physiological factors potentially underlying social facilitation of parental care. Importantly, they suggest that, even though social facilitation of parental care may increase breeding performance, it can also negatively affect other fitness components.

Feeding on ripening and over-ripening fruit: interactions between sugar, ethanol and polyphenol contents in a tropical butterfly.

In ripe fruit, energy mostly derives from sugar, while in over-ripe fruit, it also comes from ethanol. Such ripeness differences may alter the fitness benefits associated with frugivory if animals are unable to degrade ethanol when consuming over-ripe fruit. In the tropical butterfly Bicyclus anynana, we found that females consuming isocaloric solutions mimicking ripe (20% sucrose) and over-ripe fruit (10% sucrose, 7% ethanol) of the palm Astrocaryum standleyanum exhibited higher fecundity than females consuming a solution mimicking unripe fruit (10% sucrose). Moreover, relative to butterflies consuming a solution mimicking unripe fruit, survival was enhanced when butterflies consumed a solution mimicking either ripe fruit supplemented with polyphenols (fruit antioxidant compounds) or over-ripe fruit devoid of polyphenols. This suggests that (1) butterflies have evolved tolerance mechanisms to derive the same reproductive benefits from ethanol and sugar, and (2) polyphenols may regulate the allocation of sugar and ethanol to maintenance mechanisms. However, variation in fitness owing to the composition of feeding solutions was not paralleled by corresponding physiological changes (alcohol dehydrogenase activity, oxidative status) in butterflies. The fitness proxies and physiological parameters that we measured therefore appear to reflect distinct biological pathways. Overall, our results highlight that the energy content of fruit primarily affects the fecundity of B. anynana butterflies, while the effects of fruit consumption on survival are more complex and vary depending on ripening stage and polyphenol presence. The actual underlying physiological mechanisms linking fruit ripeness and fitness components remain to be clarified.

Extreme intra-clutch egg size dimorphism is not coupled with corresponding differences in antioxidant capacity and stable isotopes between eggs.

Oviparous females need to allocate resources optimally to their eggs in order to maximize their fitness. Among these resources, dietary antioxidants, acquired by females and transferred to the eggs during egg formation, can greatly affect the development and survival of the embryo and chick. In crested penguins, incubation starts after the second and last egg is laid and, as opposed to many other bird species, this egg hatches first, thereby enhancing the survival of the chick. Here, we assessed whether antioxidant and isotopic composition could underlie these differences between eggs within clutches of southern rockhopper penguins (Eudyptes chrysocome chrysocome). The second-laid egg had higher total antioxidant capacity than the first-laid egg, although this was not due to higher antioxidant concentration but to its higher mass. This suggests that resources are allocated by females at a constant rate in both eggs within clutches. Accordingly, we found a strong correlation for isotopic compositions between eggs suggesting that resources were allocated similarly to each egg within the clutch. Overall, we found little evidence for a significant role of antioxidant and isotopic compositions to explain differences in terms of embryo/chick development between eggs in crested penguins. However, since our results suggest a constant rate of antioxidant transfer from females to eggs, limiting the mass of the first-laid egg might represent a strategy for females to spare antioxidant defences and preserve self-maintenance.

Reproducing butterflies do not increase intake of antioxidants when they could benefit from them.

The significance of dietary antioxidants may be limited by the ability of animals to exploit them. However, past studies have focused on the effects of dietary antioxidants after 'antioxidant forced-feeding', and have overlooked spontaneous antioxidant intake. Here, we found that reproducing female Bicyclus anynana butterflies had higher antioxidant defences and enhanced fecundity when forced to consume antioxidants (polyphenols). Interestingly, these positive effects were not constant across the oviposition period. When given the choice between food resources with and without antioxidants, reproducing butterflies did not target antioxidants when they could have benefited the most from them. Moreover, they did not consume more antioxidants than non-reproducing butterflies. These results emphasize that, despite potential positive effects of dietary antioxidants, the ability of animals to exploit them is likely to restrict their ecological significance.

Relationships between isotopic values and oxidative status: insights from populations of gentoo penguins.

Feeding strategies can affect the balance between the production of reactive oxygen species and antioxidant defences (i.e. oxidative status). This is ecologically relevant, as variation in oxidative status can in turn strongly affect fitness. However, how animals regulate their oxidative status through their feeding behaviour under natural conditions remains poorly understood. Thus, relating the isotopic values of free-ranging animals to their oxidative status may prove useful. Here, we considered three colonies of gentoo penguins (Pygoscelis papua) in which we measured (1) δ(13)C and δ(15)N values, and (2) antioxidant defences and oxidative damage. We found that colonies with the highest δ(13)C and δ(15)N values also had the highest levels of antioxidant defences and oxidative damage, resulting in positive relationships between isotopic values and markers of oxidative status. As a result, colony segregation in terms of isotopic values was reflected by segregation in terms of oxidative markers (although more markedly for oxidative damage than for antioxidant defences). Interestingly, variation in the estimated contribution of krill in the diet of penguins followed an opposite pattern to that observed for markers of oxidative status, providing evidence that inter-population differences in terms of foraging strategies can result in inter-population differences in terms of oxidative status. More studies examining simultaneously oxidative status, isotopic signature, foraging behaviour and food allocation between parents and young are, however, needed to understand better the interplay between the foraging strategies adopted by animals in their natural habitat and their oxidative status.

Self-supplementation and effects of dietary antioxidants during acute thermal stress.

Thermal stress leads to increased production of reactive oxygen species. If an organism is not able to simultaneously mount an efficient antioxidant defense system, this may lead to increased oxidative damage, which is potentially deleterious in terms of health and fitness. Exposure to cold or heat is therefore expected to be associated with a high demand for antioxidants. In agreement, several studies have shown that supplementing the diet of thermally stressed organisms with antioxidants leads to a reduction of oxidative damage. However, whether organisms can actively supplement their diet with antioxidants to alleviate temperature-induced oxidative damage is unknown. Here, we show that captive Gouldian finches (Erythrura gouldiae) supplement their diet more with seeds rich in antioxidants below than within their thermoneutral zone. Moreover, having access to seeds rich in antioxidants at temperatures below thermoneutrality decreases their oxidative damage. These results indicate that, when facing a thermal challenge, animals are able to take advantage of the antioxidant properties of their food to improve their oxidative balance. Having access to food resources rich in antioxidants may therefore be of primary importance for organisms in their natural habitat, as it may help them to cope with oxidative constraints due to challenging temperature regimes.

The oxidative cost of unstable social dominance.

High social rank is expected to incur fitness costs under unstable social conditions. A disruption of the oxidative balance may underlie such effects, but how markers of oxidative stress vary in relation to social rank and stability is unknown. We examined in mandrills whether the mating season characterized by social instability between males (but not between females) affected their oxidative balance differently according to their social rank. Outside the mating season, high-ranking males showed the lowest levels of oxidative damage, while during the mating season, they were the only males to experience increased oxidative damage. In contrast, the mating season increased oxidative stress in all females, irrespective of their social rank. These results support the hypothesis that the coupling between social rank and social stability is responsible for differential costs in terms of oxidative stress, which may explain inter-individual differences in susceptibility to socially induced health issues.

Contrast influences female attraction to performance-based sexual signals in a songbird.

Animals do not make decisions in a bubble but often refer to previous experience when discriminating between options. Contrast effects occur when the value of a stimulus affects the response to another value of the stimulus, and the changes in value and response are in the same direction. Although contrast effects appear irrational, they could benefit decision makers when there is spatial or temporal variation and autocorrelation in the value of stimuli that elicit decisions. Here, we examined whether contrasts influence female evaluation of male performance-based sexual signals. We exposed female Lincoln's sparrows (Melospiza lincolnii) to one week of songs that we had experimentally reduced or elevated in performance, followed by a novel song of intermediate performance. We found that high-performance songs were more attractive to females than low-performance songs. Moreover, the intermediate songs were more attractive following exposure to low- than to high-performance songs. These results indicate that contrast can influence evaluation of performance-based sexual stimuli. By examining contrast effects in the ecologically relevant context of mate choice for performance, we can better understand both the adaptive value of comparative evaluation as well as the mechanisms that underlie variation in mate choice and sexual selection.

Capturing wild animal welfare: a physiological perspective.

Affective states, such as emotions, are presumably widespread across the animal kingdom because of the adaptive advantages they are supposed to confer. However, the study of the affective states of animals has thus far been largely restricted to enhancing the welfare of animals managed by humans in non-natural contexts. Given the diversity of wild animals and the variable conditions they can experience, extending studies on animal affective states to the natural conditions that most animals experience will allow us to broaden and deepen our general understanding of animal welfare. Yet, this same diversity makes examining animal welfare in the wild highly challenging. There is therefore a need for unifying theoretical frameworks and methodological approaches that can guide researchers keen to engage in this promising research area. The aim of this article is to help advance this important research area by highlighting the central relationship between physiology and animal welfare and rectify its apparent oversight, as revealed by the current scientific literature on wild animals. Moreover, this article emphasises the advantages of including physiological markers to assess animal welfare in the wild (e.g. objectivity, comparability, condition range, temporality), as well as their concomitant limitations (e.g. only access to peripheral physiological markers with complex relationships with affective states). Best-practice recommendations (e.g. replication and multifactorial approaches) are also provided to allow physiological markers to be used most effectively and appropriately when assessing the welfare of animals in their natural habitat. This review seeks to provide the foundation for a new and distinct research area with a vast theoretical and applied potential: wild animal welfare physiology.

Enhanced dispersal capacity in edge population individuals of a rapidly expanding butterfly.

Natural range shifts offer the opportunity to study the phenotypic and genetic changes contributing to colonization success. The recent range shift of the Southern small white butterfly (Pieris mannii) from the South to the North of Europe offers a prime example to examine a potential dispersal syndrome in range-expanding individuals. We compared butterflies from the core and edge populations using a multimodal approach addressing behavioral, physiological, and morphological traits related to dispersal capacity. Relative to individuals from the core range (France), individuals from the edge (Germany) showed a higher capacity and motivation to fly, and a higher flight metabolic rate. They were also smaller, which may enhance their flight maneuverability and help them cope with limited resource availability, thereby increasing their settlement success in novel environments. Altogether, the behavioral, physiological, and morphological differences observed between core and edge populations in P. mannii suggest the existence of a dispersal syndrome in range-expanding individuals. Whether these differences result from genetic and/or phenotypic responses remains, however, to be determined.