Natural populations of plants and animals face increasing levels of environmental stress due to anthropogenic activities, and there is hence a growing need to monitor these stress impacts. Biomarkers, defined as functional measures of exposure to stressors [1
], are important tools in this respect, developed and applied by ecologists for the conservation and management of biological systems. Fluctuating asymmetry (FA), which refers to small, random deviations from perfect symmetry in bilateral traits [2
], is one commonly used marker of stress. Since both sides of a bilateral trait are normally under the control of the same genome and exposed to the same environment, FA results from the inability of an individual to buffer against perturbations during its development [3
]. These perturbations act locally at a cellular level, their effects accumulating on the left and right sides separately to bring about aberrant growth and asymmetry [4
]. A high level of FA may therefore indicate exposure to stress, which led to the loss of developmental stability [5
FA as a marker of stress has been credited with being low cost, non-lethal and easy to use [6
]; moreover, it is one of the few markers where the norm (i.e., perfect symmetry) is known [8
]. It is also widely assumed to be negatively related to components of fitness such as growth and longevity [6
]. Furthermore, FA has been proposed to have a higher sensitivity to stress effects than these fitness components, making it detectable in individuals in response to stress before the corresponding decrease in fitness [9
]. It therefore has the potential to be an early warning system for the implementation of mitigation measures [10
]. However, the application of FA as a biomarker is still under much debate and controversy. While several hypotheses have been proposed, the underlying mechanisms producing developmental instability and FA are yet to be completely understood [6
]. This might be one reason why relationships between FA, stress and fitness components have often been found to be trait, stress or organism-specific [11
], with varied and often contrasting results obtained across different studies (see [12
]). FA measured on a single trait is also shown to have weak and heterogeneous relationships with stress [13
Despite these drawbacks, FA continues to be a popular indicator of stress and proxy for fitness, and has been used across various animal groups. For birds, nutritional stress during development can incur an important fitness cost; it may create energetic deficits in an individual, leading to a scenario where growth and other more vital functions are prioritized over developmental stability [14
]. Food deprivations have hence been associated with higher levels of asymmetry in various bird species [14
]. Poor diet quality with nutritive deficiencies may also lead to increased FA [17
]. As stated before, however, FA-stress relationships are not always consistent; Vangestel and Lens [18
], for instance, did not find increased FA levels in the tarsus and rectrix of House Sparrows (Passer domesticus
) under nutritional stress. Clearly, more research is needed; as correlational field studies can be confounded by uncontrolled environmental factors, experimental manipulations of stress may be a more powerful method for the study of FA.
This paper experimentally assesses the suitability of FA as a biomarker of nutritional stress, particularly in young seabirds. Food stress has become especially relevant for this group of birds in European seas in light of the recent reform of the EU Common Fisheries Policy [19
], aimed at gradually phasing out the practice of discarding bycatch by fishing vessels. This ‘discards ban’ could affect populations of scavenging seabirds that are dependent on fishery discards as a food source [20
]. Preliminary studies have suggested that lowered availability of discards is likely to produce an overall food shortage, which may result in an energetic stress in the absence of sufficient alternatives [21
]. In this context, the scavenging Lesser Black-backed Gull (Larus fuscus
) is an ideal model for the study of nutritional stress effects. This species is a generalist feeder, foraging on offshore surface prey like fish and crustaceans as well as terrestrial food and urban refuse [22
]. Individuals, however, have shown varying degrees of specialisation in their feeding habits [23
], which means that these dietary shifts and restrictions are likely to have a spectrum of effects across individuals.
An aviary experiment was conducted during the 2015 breeding season where newly hatched L. fuscus chicks were reared on a range of diets expected to induce different degrees of nutritional stress through their different energetic values. Fluctuating asymmetry was then assessed for each chick at the end of the experiment by measuring bilateral differences in three morphological traits: the tarsus, wing and primary feather, analysed both independently and combined as multi-trait FA. The low energy diets were expected to constitute significant levels of nutritional stress that impair chick development. We aimed to assess if fluctuating asymmetry across the three measured traits consistently reflected the nutritional stress exposure in developing L. fuscus chicks.
The central hypothesis tested in our study was that increased levels of nutritional stress (induced by energetic constraints) would result in more asymmetric growth at the organism-wide level. Chicks of the most stressed treatment (S3) did indeed have the highest levels of FA overall (Figure 2
), and statistical analysis showed that FA was associated positively with stress for the feather, in accordance with our predictions. However, we were unable to establish conclusive, linear relationships between stress and FA for the wings, tarsi and multi-trait estimates. Lack of concordance between traits was further seen in the absence of between-trait correlations in unsigned FA values. Therefore, while the degree of fluctuating asymmetry does appear to reflect energetic stress exposure in developing chicks, FA-stress relationships were not uniform across traits.
The tarsus is a relatively well-studied trait for fluctuating asymmetry in birds, and has been previously linked to a number of different stressors (see [38
] for examples). The similar levels of tarsus FA between our stress treatments (Figure 2
), and its mixed and mostly non-significant relationships with stress across the different hatching periods (Table 3
) are therefore surprising and contrary to expectations. Furthermore, although wing FA showed a positive relationship with stress, confidence intervals indicate that this relationship is unreliable (Table 4
). This heterogeneity between traits likely resulted in the failure to detect any FA-stress relationships using the multi-trait approach. Trait-specific asymmetry patterns are in agreement with previous findings (see [11
]) and may be attributed to several (non-mutually exclusive) causes.
First and foremost, different traits can have variable responses to stress. For instance, the compensational growth hypothesis (sensu [40
]) proposes that the stage of ontogeny at which a trait is exposed to stress can influence the manifestation of FA across it. According to this hypothesis, regulatory mechanisms between the two sides of a trait work to restore trait symmetry towards the end of development. Among the studied traits, the tarsi were fully grown (or close to) in most chicks at the time of measurement, which means that compensational growth could have restored their bilateral symmetry. Conversely, the wings and primary feathers were still developing at the end of our experiment and hence would have been more likely to be asymmetrical. However, compensational growth is just one of the mechanisms proposed to describe the ontogeny of FA, and many studies have found evidence contrary to it (see [41
] for examples).
Similarly, heterogeneous trait responses may be explained by the concept of “windows of opportunity” [43
]. A trait is said to be vulnerable to environmental stressors only during certain developmental stages, leading to the production of visible aberrant phenotypes. These windows of opportunity may vary among traits [43
]; the tarsi and wings may have experienced only restricted levels of energetic stress during the vulnerable period of their development. However, given that stress exposure was nearly constant throughout our experiment, this is not a very likely explanation of our results. Alternatively, Aparicio and Bonal [45
] proposed that the structural composition of a trait may affect relationships between stress and FA as well. In particular, traits with more structural components per unit length may be less likely to manifest fluctuating asymmetry.
Trait-specific susceptibility to FA may also be related to the functional importance of a trait [10
]. Traits of higher functional value should be subjected to stronger stabilizing selection, as minor reductions in their symmetry can impose a substantial fitness cost. These will therefore be less susceptible to developmental perturbations [10
]. For instance, Gummer and Brigham [47
] found lower degrees of asymmetry in traits having a higher role in mobility in Little Brown Bats (Myotis lucifugus
). All traits measured in our study were related to mobility, i.e., tarsi for walking and wing and feathers for flight. However, the primary feathers and wings may have been of relatively lower functional importance in young chicks yet to fledge, as compared to tarsi. If so, the latter can be expected to be more strongly buffered against the nutritional stress, hence weakening the FA-stress relationships. Furthermore, Boonekamp et al. [48
] have suggested that functional traits that are sensitive to developmental conditions may still be poor fitness predictors, given that their development is likely to be more strongly canalized.
Finally, inconsistent results may arise from problems with statistical analysis or experimental design. We acknowledge that our sample sizes (Figure 2
) were on the low side for study of relationships with fluctuating asymmetry, and the latter should therefore be interpreted with some caution. In particular, the feather sample size of the S3 treatment was very small (three individuals) as compared to the other groups, which may have biased the analysis of this trait. However, we believe that, overall, our statistical analyses were biologically and statistically sound. FA variance components were significantly larger than the error components in the mixed model estimation (Table 1
), indicating the absence of measurement bias. This model also accounted for directional asymmetry, which may otherwise confound the FA estimation [29
]. Our results are also unlikely to be compromised by experimental flaws, total energy ingestions and body sizes of the chicks (Figure 1
) verify that our treatments were sufficient to induce different stress levels. Moreover, being an experimental study, it is less likely to be confounded by unknown environmental factors. Therefore, the heterogeneous FA-stress relationships in our results appear to be at least partly real, rather than artefacts.