Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Stress response varies with plumage colour and local habitat in feral pigeons

International audienceBird populations exposed to different extrinsic conditions often differ in the responsiveness of the hypothalamo–pituitary–adrenal (HPA) axis and thus in corticosterone response that individuals mount when facing stressful events. However, the contribution of genetic variation to among-individual variability in HPA axis responsiveness across different environmental conditions is poorly understood. Melanin-based coloured types provide reliable phenotypic markers of alternative genotypes underlying stress coping styles. Large variations in melanin-based colouration are heritable in feral pigeons. We tested whether melanin-based colouration is associated with variation in corticosterone stress response in feral pigeons. To this end, we examined how corticosterone response varies both within and between differently coloured individuals across different environmental conditions. Differently coloured individuals produced different stress-induced corticosterone levels in relation to their environmental conditions: dark pigeons exhibited a higher corticosterone when originating from rural habitats, while this was not observed in pale pigeons. This suggests that among-population variation in stress response is higher in dark pigeons, this variation possibly reflecting adjustment and/or (epi)genetic adaptation to environmental conditions. In addition, corticosterone response increased with the degree of melanin-based colouration in pigeons originating from rural habitats but not in pigeons originating from more urbanized populations, resulting in the coexistence of alternative stress responses in some populations, but not in others. Our results suggest that species with melanin-based variation in differently urbanized populations along rural–urban gradients are potentially good candidate systems for studying stress coping styles under alternative selective regimes

The multivariate egg: quantifying within- and among-clutch correlations between maternally derived yolk immunoglobulins and yolk androgens using multivariate mixed models

Egg components are important mediators of prenatal maternal effects in birds and other oviparous species. Because different egg components can have opposite effects on offspring phenotype, selection is expected to favour their mutual adjustment, resulting in a significant covariation between egg components within and / or among clutches. Here we tested for such correlations between maternally-derived yolk immunoglobulins and yolk androgens in great tit (Parus major) eggs using a multivariate mixed model approach. We found no association between yolk immunoglobulins and yolk androgens within clutches, indicating that within clutches the two egg components are deposited independently. Across clutches, however, there was a significant negative relationship between yolk immunoglobulins and yolk androgens, suggesting that selection has co-adjusted their deposition. Furthermore, an experimental manipulation of ectoparasite load affected patterns of covariance among egg components. Yolk immunoglobulins are known to play an important role in nestling immune defence shortly after hatching, whereas yolk androgens, although having growth-enhancing effects under many environmental conditions, can be immunosuppressive. We therefore speculate that variation in the risk of parasitism may play an important role in shaping optimal egg composition and may lead to the observed pattern of yolk immunoglobulin and yolk androgen deposition across clutches. More generally, our case study exemplifies how multivariate mixed model methodology presents a flexible tool to not only quantify, but also test patterns of (co)variation across different organisational levels and environments, allowing for powerful hypothesis testing in ecophysiology