Evaluating tramway infrastructure on biodiversity and ecosystem services.

Tramways in urban areas for mass transit has been suggested to have a lower environmental footprint than roads. However, studies on the impact of tramways and the surrounding infrastructure on biodiversity is extremely rare despite the potential ecological effects associated with this anthropogenic feature. Surprisingly, we found fewer than 10 papers published on tramway-wildlife interactions, which is significantly lower (vs dozens of thousands) than that of other transportation methods. As tramways and stations may be managed sustainably by planting short vegetation on the track and roofs of tramway stations, they may be good examples of land-sharing policies in green urban planning, improving both biodiversity and people's well-being. The potential environmental benefits of green practices for commercially available tramways should be strictly tested and applied, especially in the context of the growing popularity of tramway systems worldwide.

Avian colouration in a polluted world: a meta-analysis.

Brilliant, diverse colour ornaments of birds were one of the crucial cues that led Darwin to the idea of sexual selection. Although avian colouration plays many functions, including concealment, thermoregulation, or advertisement as a distasteful prey, a quality-signalling role in sexual selection has attracted most research attention. Sexually selected ornaments are thought to be more susceptible to external stressors than naturally selected traits, and as such, they might be used as a test for environmental quality. For this reason, the last two decades have seen numerous studies on the impact of anthropogenic pollution on the expression of various avian colour traits. Herein, we provide the first meta-analytical summary of these results and examine whether there is an interaction between the mechanism of colour production (carotenoid-based, melanin-based and structural) and the type of anthropogenic factor (categorised as heavy metals, persistent organic pollutants, urbanisation, or other). Following the assumption of heightened condition dependence of ornaments under sexual selection, we also expected the magnitude of effect sizes to be higher in males. The overall effect size was close to significance and negative, supporting a general detrimental impact of anthropogenic pollutants on avian colouration. In contrast to expectations, there was no interaction between pollution types and colour-producing mechanisms. Yet there were significant differences in sensitivity between colour-producing mechanisms, with carotenoid-based colouration being the most affected by anthropogenic environmental disturbances. Moreover, we observed no significant tendency towards heightened sensitivity in males. We identified a publication gap on structural colouration, which, compared to pigment-based colouration, remains markedly understudied and should thus be prioritised in future research. Finally, we call for the unification of methods used in colour quantification in ecological research to ensure comparability of results among studies.

What do zebra finches learn besides singing? Systematic mapping of the literature and presentation of an efficient associative learning test.

The process of learning in birds has been extensively studied, with a focus on species such as pigeons, parrots, chickens, and crows. In recent years, the zebra finch has emerged as a model species in avian cognition, particularly in song learning. However, other cognitive domains such as spatial memory and associative learning could also be critical to fitness and survival, particularly during the intensive juvenile period. In this systematic review, we provide an overview of cognitive studies on zebra finches, with a focus on domains other than song learning. Our findings indicate that spatial, associative, and social learning are the most frequently studied domains, while motoric learning and inhibitory control have been examined less frequently over 30 years of research. All of the 60 studies included in this review were conducted on captive birds, limiting the generalizability of the findings to wild populations. Moreover, only two of the studies were conducted on juveniles, highlighting the need for more research on this critical period of learning. To address this research gap, we propose a high-throughput method for testing associative learning performance in a large number of both juvenile and adult zebra finches. Our results demonstrate that learning can occur in both age groups, thus encouraging researchers to also perform cognitive tests on juveniles. We also note the heterogeneity of methodologies, protocols, and subject exclusion criteria applied by different researchers, which makes it difficult to compare results across studies. Therefore, we call for better communication among researchers to develop standardised methodologies for studying each cognitive domain at different life stages and also in their natural conditions.

Canalised and plastic components of melanin-based colouration: a diet-manipulation experiment in house sparrows.

Whether melanin-based plumage colouration accurately reflects a bird's quality is still controversial. To better understand potential mechanisms behind the observed variation in plumage colouration, we shifted our attention from a high-level expression of colour to low-level physiological phenomena by targeting the microstructure and pigment content of the feather. In a well-studied model system, the house sparrow (Passer domesticus), we combined an experimental manipulation of birds' physiological condition and availability of resources that are key to the production of the studied colouration (phenylalanine and tyrosine (PT). We found that feathers from sparrows fed with the control diet had noticeably lower values of brightness, suggesting a higher quality of the ornamental "blackness" in comparison to those sampled from birds fed with a PT-reduced diet. Electron paramagnetic resonance (EPR) spectroscopy detected higher melanin concentrations in samples from the control than the PT-reduced group. Our multi-level analysis excluded mechanisms such as barbule density and melanosomes' distribution, clearly pointing to the finest-level proxy of colour: the concentration of melanin in melanosomes themselves. Despite melanins being manufactured by birds endogenously, the efficiency of melanogenesis can be noticeably limited by diet. As a result, the birds' plumage colouration is affected, which may entail consequences in social signalling.

Concerted evolution of body mass, cell size and metabolic rate among carabid beetles.

Alterations in cell number and size are apparently associated with the body mass differences between species and sexes, but we rarely know which of the two mechanisms underlies the observed variance in body mass. We used phylogenetically informed comparisons of males and females of 19 Carabidae beetle species to compare body mass, resting metabolic rate, and cell size in the ommatidia and Malpighian tubules. We found that the larger species or larger sex (males or females, depending on the species) consistently possessed larger cells in the two tissues, indicating organism-wide coordination of cell size changes in different tissues and the contribution of these changes to the origin of evolutionary and sex differences in body mass. The species or sex with larger cells also exhibited lower mass-specific metabolic rates, and the interspecific mass scaling of metabolism was negatively allometric, indicating that large beetles with larger cells spent relatively less energy on maintenance than small beetles. These outcomes also support existing hypotheses about the fitness consequences of cell size changes, postulating that the low surface-to-volume ratio of large cells helps decrease the energetic demand of maintaining ionic gradients across cell membranes. Analyses with and without phylogenetic information yielded similar results, indicating that the observed patterns were not biased by shared ancestry. Overall, we suggest that natural selection does not operate on each trait independently and that the linkages between concerted cell size changes in different tissues, body mass and metabolic rate should thus be viewed as outcomes of correlational selection.

Food consumption in ground beetles is limited under hypoxic conditions in response to ad libitum feeding, but not restricted feeding.

Habitats on land with low oxygen availability provide unique niches inhabited by numerous species. The occupation of such hypoxic niches by animals is hypothesized to come at a cost linked to the limitations of aerobic metabolism and thus energy budget but may also provide benefits through physical protection from predators and parasitoids or reduced competition for food. We investigated the effects of hypoxic conditions on standard metabolic rate (SMR) and specific dynamic action (SDA) in male Carabus nemoralis. SMR and SDA were determined under three manipulated oxygen availabilities: 7, 14 and 21% O2 and two feeding regimes: limited or ad libitum food consumption. In both hypoxic conditions, C. nemoralis was able to maintain SMR at levels similar to those in normoxia. When the meal size was limited, SDA duration did not differ among the oxygen availability conditions, but SDA was smaller under hypoxic conditions than at normoxic levels. The relative cost of digestion was significantly higher in normoxia than in hypoxia, but it did not affect net energy intake. In contrast, when offered a large meal to simulate ad libitum food conditions, beetles reduced their food consumption and net energy gain by 30% under hypoxia. Oxygen availability may influence the consumed prey size: the hypoxic condition did not limit net energy gain when the beetles fed on a small meal but did when they fed on a large meal. The results indicate that meal size is an important variable in determining differences in physiological costs and whole animal energy budgets at different concentrations of environmental oxygen levels.

Effects of fat and exoskeletal mass on the mass scaling of metabolism in Carabidae beetles.

The rate at which organisms metabolize resources and consume oxygen is tightly linked to body mass. Typically, there is a sub-linear allometric relationship between metabolic rates and body mass (mass-scaling exponent b < 1). The origin of this pattern remains one of the most intriguing and hotly debated topics in evolutionary physiology. A decrease in mass-specific metabolic rates in larger organisms might reflect disproportionate increases in body components with low metabolic activity, such as storage and skeletal tissues. Addressing this hypothesis, we studied standard metabolic rates, body mass, and fat and exoskeletal mass in males and females from 15 species of Carabidae beetles. There was a sub-linear allometric relationship of metabolic rate with body mass: b = 0.72 (phylogeny not considered), b = 0.54 (phylogeny considered). The latter exponent was significantly lower than 0.75, which is sometimes regarded as a universal exponent value in the mass scaling of metabolic rates. Contrary to our hypothesis, the relative contribution of fat and the exoskeleton to body mass decreased, rather than increased with body mass, as indicated by the sub-linear allometric mass scaling of both components (b < 1). Supporting the role of metabolically inert body components in shaping metabolic scaling, the exponents (b) for metabolism became slightly smaller (b = 0.70, phylogeny not considered; 0.52, phylogeny considered) when we removed lipids and the exoskeleton from body mass calculations and considered only the lean mass of soft tissue in the mass scaling. Overall, our results indicate that, in beetles, the relative content of metabolically inert components changes across species according to species-specific body mass. Nevertheless, we did not find evidence that this changing contribution plays a central role in the origin of interspecific metabolic scaling in carabids. Our findings stress the need for finding alternative explanations, at least in carabids, for the origin of the mass scaling of metabolic rates.

Physical mechanism or evolutionary trade-off? Factors dictating the relationship between metabolic rate and ambient temperature in carabid beetles.

The tight association between ambient temperature (T) and metabolic rate (MR) is a common occurrence in ectotherms, but the determinants of this association are not fully understood. This study examined whether the relationship between MR and T is the same among individuals, as predicted by the Universal Temperature Dependence hypothesis, or whether this relationship differs between them. We used flow-through respirometry to measure standard MR and to determine gas exchange patterns for 111 individuals of three Carabidae species which differ in size (Abax ovalis, Carabus linnei and C. coriaceus), exposed to four different temperatures (ten individuals of each species measured at 6, 11, 16 and 21°C). We found a significant interaction between ln body mass and the inverse of temperature, indicating that in a given species, the effect of temperature on MR was weaker in larger individuals than in smaller individuals. Overall, this finding shows that the thermal dependence of MR is not body mass invariant. We observed three types of gas exchange patterns among beetles: discontinuous, cyclic and continuous. Additionally, the appearance of these patterns was associated with MR and T. Evolution in diverse terrestrial environments could affect diverse ventilation patterns, which accommodate changes in metabolism in response to temperature variation. In conclusion, explaining the variance in metabolism only through fundamental physical laws of thermodynamics, as predicted by the Universal Temperature Dependence hypothesis, appears to oversimplify the complexity of nature, ignoring evolutionary trade-offs that should be taken into account in the temperature - metabolism relationship.

Mass scaling of metabolic rates in carabid beetles (Carabidae) - the importance of phylogeny, regression models and gas exchange patterns.

The origin of the allometric relationship between standard metabolic rate (MR) and body mass (M), often described as MR=aMb , remains puzzling, and interpretation of the mass-scaling exponent, b may depend on the methodological approach, shapes of residuals, coefficient of determination (r2) and sample size. We investigated the mass scaling of MRs within and between species of Carabidae beetles. We used ordinary least squares (OLS) regression, phylogenetically generalized least squares (PGLS) regression and standardized major axis (SMA) regression to explore the effects of different model-fitting methods and data clustering caused by phylogenetic clades (grade shift) and gas exchange patterns (discontinuous, cyclic and continuous). At the interspecific level, the relationship between MR and M was either negatively allometric (b<1) or isometric (b=1), depending on the fitting method. At the intraspecific level, the relationship either did not exist or was isometric or positively allometric (b>1), and the fit was significantly improved after the analyzed dataset was split according to gas exchange patterns. The studied species originated from two distinct phylogenetic clades that had different intercepts but a common scaling exponent (OLS, 0.61) that was much shallower than the scaling exponent for the combined dataset for all species (OLS, 0.71). The best scaling exponent estimates were obtained by applying OLS while accounting for grade shifts or by applying PGLS. Overall, we show that allometry of MR in insects can depend heavily on the model fitting method, the structure of phylogenetic non-independence and ecological factors that elicit different modes of gas exchange.

The closed spiracle phase of discontinuous gas exchange predicts diving duration in the grasshopper Paracinema tricolor.

The discontinuous gas exchange (DGE) pattern of respiration shown by many arthropods includes periods of spiracle closure (C-phase) and is largely thought to serve as a physiological adaptation to restrict water loss in terrestrial environments. One major challenge to this hypothesis is to explain the presence of DGE in insects in moist environments. Here, we show a novel ecological correlate of the C-phase, namely, diving behaviour in mesic Paracinema tricolor grasshoppers. Notably, maximal dive duration is positively correlated with C-phase length, even after accounting for mass scaling and absolute metabolic rate. Here, we propose that an additional advantage of DGE may be conferred by allowing the tracheal system to act as a sealed underwater oxygen reservoir. Spiracle closure may facilitate underwater submersion, which, in turn, may contribute to predator avoidance, the survival of accidental immersion or periodic flooding and the exploitation of underwater resources.

Hold your breath beetle-Mites!

Respiratory gas exchange in insects occurs via a branching tracheal system. The entrances to the air-filled tracheae are the spiracles, which are gate-like structures in the exoskeleton. The open or closed state of spiracles defines the three possible gas exchange patterns of insects. In resting insects, spiracles may open and close over time in a repeatable fashion that results in a discontinuous gas exchange (DGE) pattern characterized by periods of zero organism-to-environment gas exchange. Several adaptive hypotheses have been proposed to explain why insects engage in DGE, but none have attracted overwhelming support. We provide support for a previously untested hypothesis that posits that DGE minimizes the risk of infestation of the tracheal system by mites and other agents. Here, we analyze the respiratory patterns of 15 species of ground beetle (Carabidae), of which more than 40% of individuals harbored external mites. Compared with mite-free individuals, infested one's engaged significantly more often in DGE. Mite-free individuals predominantly employed a cyclic or continuous gas exchange pattern, which did not include complete spiracle closure. Complete spiracle closure may prevent parasites from invading, clogging, or transferring pathogens to the tracheal system or from foraging on tissue not protected by thick chitinous layers.

Automated measurement of ommatidia in the compound eyes of beetles.

The size of the ommatidia that compose the insect compound eye is linked to visual capacity, physiological performance, and cell size. Therefore, rapid and reliable methods for measuring ommatidia can advance research on insect ecology and evolution. We developed an automated method to measure ommatidia in nail polish imprints of the eyes of three Carabidae beetle species using the widely available, free software ImageJ. Our automated method was equivalent to a traditional manual method in terms of accuracy but had the advantage of being 70 times faster. We provide access to our algorithm, which can be used to investigate biological phenomena ranging from the functional architecture of the compound eye to the cellular basis of the evolution of body size and metabolic rates.

The Janus face of α-toxin: a potent mediator of cytoprotection in staphylococci-infected macrophages.

After phagocytosis by macrophages, Staphylococcus aureus evades killing in an α-toxin-dependent manner, and then prevents apoptosis of infected cells by upregulating expression of antiapoptotic genes like MCL-1 (myeloid cell leukemia-1). Here, using purified α-toxin and a set of hla-deficient strains, we show that α-toxin is critical for the induction of MCL-1 expression and the cytoprotection of infected macrophages. Extracellular or intracellular treatment of macrophages with α-toxin alone did not induce cytoprotection conferred by increased Mcl-1, suggesting that the process is dependent on the production of α-toxin by intracellular bacteria. The increased expression of MCL-1 in infected cells was associated with enhanced NFκB activation, and subsequent IL-6 secretion. This effect was only partially inhibited by blocking TLR2, which suggests the participation of intracellular receptors in the specific recognition of S. aureus strains secreting α-toxin. Thus, S. aureus recognition by intracellular receptors and/or activation of downstream pathways leading to Mcl-1 expression is facilitated by α-toxin released by intracellular bacteria which permeabilize phagosomes, ensuring pathogen access to the cytoplasmatic compartment. Given that the intracellular survival of S. aureus depends on α-toxin, we propose a novel role for this agent in the protection of the intracellular niche, and further dissemination of staphylococci by infected macrophages.

The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages.

As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.

Phagocytosis of Staphylococcus aureus by macrophages exerts cytoprotective effects manifested by the upregulation of antiapoptotic factors.

It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection.

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages.

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.