A blood-based multi-biomarker approach reveals different physiological responses of common kestrels to contrasting environments.

The increase of urbanization and agricultural activities is causing a dramatic reduction of natural environments. As a consequence, animals need to physiologically adjust to these novel environments, in order to exploit them for foraging and breeding. The aim of this work was to compare the physiological status among nestling common kestrels (Falco tinnunculus) that were raised in nest-boxes located in more natural, rural, or urban areas in a landscape with a mosaic of land uses around Rome in Central Italy. A blood-based multi-biomarker approach was applied to evaluate physiological responses at multiple levels, including antioxidant concentrations, immunological functions, genotoxicity, and neurotoxicity. We found lower concentrations of glutathione and GSH:GSSG ratio values and higher proportions of monocytes in urban birds compared to the other areas. We also found higher DNA damage in rural compared to urban and natural krestels and inhibition of butyrylcholinesterase activity in urban and natural birds compared to rural area. Finally, we found similar values among study areas for respiratory burst, complement system, bactericidal capacity, and plasma non-enzymatic antioxidant capacity. These results suggest that (i) city life does not necessarily cause physiological alterations in kestrels compared to life in other habitats, and (ii) environmental pressures are likely to differ in typology and intensity across habitats requiring specific responses that a multi-biomarker approach can help to detect. Further studies are needed to assess which factors are responsible for the physiological differences among city, rural, and natural birds, and whether these differences are consistent across time and space.

Vultures respond to challenges of near-ground thermal soaring by varying bank angle.

Many large birds rely on thermal soaring flight to travel cross-country. As such, they are under selective pressure to minimise the time spent gaining altitude in thermal updrafts. Birds should be able to maximise their climb rates by maintaining a position close to the thermal core through careful selection of bank angle and airspeed; however, there have been few direct measurements of either parameter. Here, we apply a novel methodology to quantify the bank angles selected by soaring birds using on-board magnetometers. We couple these data with airspeed measurements to parameterise the soaring envelope of two species of Gyps vulture, from which it is possible to predict 'optimal' bank angles. Our results show that these large birds respond to the challenges of gaining altitude in the initial phase of the climb, where thermal updrafts are weak and narrow, by adopting relatively high, and conserved, bank angles (25-35 deg). The bank angle decreased with increasing altitude, in a manner that was broadly consistent with a strategy of maximising the rate of climb. However, the lift coefficients estimated in our study were lower than those predicted by theoretical models and wind-tunnel studies. Overall, our results highlight how the relevant currency for soaring performance changes within individual climbs: when thermal radius is limiting, birds vary bank angle and maintain a constant airspeed, but speed increases later in the climb in order to respond to decreasing air density.

Long necks enhance and constrain foraging capacity in aquatic vertebrates.

Highly specialized diving birds display substantial dichotomy in neck length with, for example, cormorants and anhingas having extreme necks, while penguins and auks have minimized necks. We attached acceleration loggers to Imperial cormorants Phalacrocorax atriceps and Magellanic penguins Spheniscus magellanicus, both foraging in waters over the Patagonian Shelf, to examine the difference in movement between their respective heads and bodies in an attempt to explain this dichotomy. The penguins had head and body attitudes and movements that broadly concurred throughout all phases of their dives. By contrast, although the cormorants followed this pattern during the descent and ascent phases of dives, during the bottom (foraging) phase of the dive, the head angle differed widely from that of the body and its dynamism (measured using vectorial dynamic acceleration) was over four times greater. A simple model indicated that having the head on an extended neck would allow these cormorants to half the energy expenditure that they would expend if their body moved in the way their heads did. This apparently energy-saving solution is likely to lead to greater heat loss though and would seem tenable in slow-swimming species because the loss of streamlining that it engenders would make it detrimental for fast-swimming taxa such as penguins.

Large-scale navigational map in a mammal.

Navigation, the ability to reach desired goal locations, is critical for animals and humans. Animal navigation has been studied extensively in birds, insects, and some marine vertebrates and invertebrates, yet we are still far from elucidating the underlying mechanisms in other taxonomic groups, especially mammals. Here we report a systematic study of the mechanisms of long-range mammalian navigation. High-resolution global positioning system tracking of bats was conducted here, which revealed high, fast, and very straight commuting flights of Egyptian fruit bats (Rousettus aegyptiacus) from their cave to remote fruit trees. Bats returned to the same individual trees night after night. When displaced 44 km south, bats homed directly to one of two goal locations--familiar fruit tree or cave--ruling out beaconing, route-following, or path-integration mechanisms. Bats released 84 km south, within a deep natural crater, were initially disoriented (but eventually left the crater toward the home direction and homed successfully), whereas bats released at the crater-edge top homed directly, suggesting navigation guided primarily by distal visual landmarks. Taken together, these results provide evidence for a large-scale "cognitive map" that enables navigation of a mammal within its visually familiar area, and they also demonstrate the ability to home back when translocated outside the visually familiar area.

Blood transcriptome analysis of common kestrel nestlings living in urban and non-urban environments.

Urbanisation is one of the main anthropogenic forms of land cover affecting an ever-increasing number of wild animals and their habitats. Physiological plasticity represents an important process through which animals can adjust to the novel conditions of anthropogenic environments. Relying on the analysis of gene expression, it is possible to identify the molecular responses to the habitat conditions and infer possible environmental factors that affect the organismal physiology. We have quantified for the first time the blood transcriptome of common kestrel (Falco tinnunculus) nestlings living in urban sites and compared it to the transcriptome of kestrel nestlings inhabiting rural and natural environments. We found mild differences in the expression of genes among sites, indicating adaptability or acclimation of the birds to the urban habitat. We identified 58 differentially expressed genes between urban and natural kestrels, and 12 differentially expressed genes between urban and rural kestrels. The most striking differences among sites involved inflammatory-immunological, metabolic, apoptosis, DNA repair and development genes. In particular, we found that (i) urban kestrel nestlings had higher expression of genes linked to inflammation, repair of DNA damage, or apoptosis than natural kestrel nestlings, and (ii) natural and rural kestrel nestlings had higher expression of genes linked to the development and activation of immune cells, type I interferon response, or major histocompatibility complex than urban kestrel nestlings. Finally, the KEGG enrichment analysis identified the insulin signalling as the main pathway that differed between natural and urban kestrel nestlings. This is one of a limited number of studies on vertebrates that revealed habitat-associated differences in the transcriptome. It paves the way for further in-depth studies on the links between physiological variation and habitat structure at different spatial and temporal scales.

Analysis of organochlorines and polycyclic aromatic hydrocarbons designed for pollutant biomonitoring in three seabird matrices.

Pollutant biomonitoring demands analytical methods to cover a wide range of target compounds, work with minimal sample amounts, and apply least invasive and reproducible sampling procedures. We developed a method to analyse 68 bioaccumulative organic pollutants in three seabird matrices: plasma, liver, and stomach oil, representing different exposure phases. Extraction efficiency was assessed based on recoveries of spiked surrogate samples, then the method was applied to environmental samples collected from Scopoli's shearwater (Calonectris diomedea). Extraction was performed in an ultrasonic bath, purification with Florisil cartridges (5 g, 20 mL), and analysis by GC-Orbitrap-MS. Quality controls at 5 ng yielded satisfactory recoveries (80-120%) although signal intensification was found for some compounds. The method permitted the detection of 28 targeted pollutants in the environmental samples. The mean sum of organic pollutants was 4.25 ± 4.83 ng/g in plasma, 1634 ± 2990 ng/g in liver, and 233 ± 111 ng/g in stomach oil (all wet weight). Pollutant profiles varied among the matrices, although 4,4'-DDE was the dominant compound overall. This method is useful for pollutant biomonitoring in seabirds and discusses the interest of analysing different matrices.

High-Performance PCR for Alleles Discrimination of Chromo-Helicase-DNA Binding Protein (CHD1) Gene in Bird Sexing.

Genetic analyses aiming at assessing the presence of specific sequences or alleles are often carried out by PCR. Sexing of most birds is nowadays based on PCR with "universal" primers and relies on the assessment of the presence of the sex-linked CHD1-Z and -W alleles. The entire workflow is relatively time-consuming, especially for batch analyses, whereas methods that allow carrying out the entire procedure in a short time are highly desirable. The only method for outdoor analyses reported so far relies on LAMP; however; it fails to work properly in Procellariiformes. Besides improving the LAMP test; we have developed a PCR-based DNA amplification procedure (named high-performance PCR); whose unique features allow it to outperform standard PCR; making possible the direct, in-tube visual reading of results. We tested it with specifically designed Procellariiformes-targeted primer sets for rapid sexing of the birds using fluorimetric detection. The protocol, combined with rapid DNA extraction, allows for fast reading of results without electrophoresis within less than 1 h from sampling. The technique could be extended to other species, as well as to many other applications.

Individual variability in diving behavior of the Black-vented Shearwater in an ever-changing habitat.

Oceanic mesoscale systems are characterized by inherent variability. Climatic change adds entropy to this system, making it a highly variable environment in which marine species live. Being at the higher levels of the food chain, predators maximize their performance through plastic foraging strategies. Individual variability within a population and the possible repeatability across time and space may provide stability in a population facing environmental changes. Therefore, variability and repeatability of behaviors, particularly diving behavior, could play an important role in understanding the adaptation pathway of a species. This study focuses on characterizing the frequency and timing of different dives (termed simple and complex) and how these are influenced by individual and environmental characteristics (sea surface temperature, chlorophyll a concentration, bathymetry, salinity, and Ekman transport). This study is based on GPS and accelerometer-recorded information from a breeding group of 59 Black-vented Shearwater and examine consistency in diving behavior at both individual and sex levels across four different breeding seasons. The species was found to be the best performing free diver in the Puffinus genus with a maximum dive duration of 88 s. Among the environmental variables assessed, a relationship was found with active upwelling conditions enhancing low energetic cost diving, on the contrary, reduced upwelling and warmer superficial waters induce more energetically demanding diving affecting diving performance and ultimately body conditions. The body conditions of Black-vented Shearwaters in 2016 were worse than in subsequent years, in 2016, deepest and longest complex dives were recorded, while simple dives were longer in 2017-2019. Nevertheless, the species' plasticity allows at least part of the population to breed and feed during warmer events. While carry-over effects have already been reported, the effect of more frequent warm events is still unknown.

Tracking the exposure of a pelagic seabird to marine plastic pollution.

We aimed to describe how debris originated from coastal cities and fisheries circulates and accumulates along the Argentine continental shelf and its potential interaction with southern giant petrels (SGP, Macronectes giganteus). We used tracking data of 31 SGPs (adults and juveniles) from Patagonian colonies. Lagrangian simulations of particles were released from coastal cities and fisheries. Oceanographic features together with plastic input generated a corridor of debris through the Argentine shelf with areas of high debris accumulation, exposing SGP to plastic consumption. During chick provisioning trips 93.9% of petrel's locations overlapped with areas of plastic accumulation. Although early developmental stages were more exposed to particles from cities, the exposure of petrels (all classes) to debris from fisheries was 10% higher than from cities. Measures to reduce debris from fisheries, would reduce plastic ingestion by giant petrels. Proper management of open sky dumpsters would reduce plastic consumption by chicks and juveniles.

Year-round variation in the isotopic niche of Scopoli's shearwater (Calonectris diomedea) breeding in contrasting sea regions of the Mediterranean Sea.

Top marine predators are key components of marine food webs. Among them, long-distance migratory seabirds, which travel across different marine ecosystems over the year, may experience important year-round changes in terms of oceanographic conditions and availability of trophic resources. We tested whether this was the case in the Scopoli's shearwater (Calonectris diomedea), a trans-equatorial migrant and top predator, by sampling birds breeding in three environmentally different regions of the Mediterranean Sea. The analysis of positional data and stable isotopes (δ1³C and δ15N) of target feathers revealed that birds from the three regions were spatially segregated during the breeding period while they shared non-breeding areas in the Atlantic Ocean. Isotopic baseline levels of N and C (meso-zooplankton) were significantly different among marine regions during breeding. Such variation was reflected at the higher trophic levels of pelagic and demersal fish muscles as well as in shearwater feathers grown in the Mediterranean. δ15N- and δ13C-adjusted values of shearwaters were significantly different among populations suggesting that birds from different breeding areas relied on prey species from different trophic levels. Conversely, the non-breeding spatial and isotopic niches overlapped greatly among the three populations. Shearwater trophic niches during breeding were narrower and segregated compared to the non-breeding period, revealing a high plasticity in trophic resource use. Overall, this study highlights seasonal and region-specific use of trophic resources by Scopoli's shearwater, suggesting a broad trophic plasticity and possibly a high adaptability to environmental changes.

Fine-scale changes in speed and altitude suggest protean movements in homing pigeon flights.

The power curve provides a basis for predicting adjustments that animals make in flight speed, for example in relation to wind, distance, habitat foraging quality and objective. However, relatively few studies have examined how animals respond to the landscape below them, which could affect speed and power allocation through modifications in climb rate and perceived predation risk. We equipped homing pigeons (Columba livia) with high-frequency loggers to examine how flight speed, and hence effort, varies in relation to topography and land cover. Pigeons showed mixed evidence for an energy-saving strategy, as they minimized climb rates by starting their ascent ahead of hills, but selected rapid speeds in their ascents. Birds did not modify their speed substantially in relation to land cover, but used higher speeds during descending flight, highlighting the importance of considering the rate of change in altitude before estimating power use from speed. Finally, we document an unexpected variability in speed and altitude over fine scales; a source of substantial energetic inefficiency. We suggest this may be a form of protean behaviour adopted to reduce predation risk when flocking is not an option, and that such a strategy could be widespread.

Prion protein amino acid determinants of differential susceptibility and molecular feature of prion strains in mice and voles.

The bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases.

Patterns of mercury exposure and relationships with isotopes and markers of oxidative status in chicks of a Mediterranean seabird.

The Mediterranean basin is a hotspot of mercury (Hg) contamination owing to intense anthropogenic emissions, volcanic activity and oligotrophic conditions. Little work has been done to assess the sources of Hg exposure for seabirds and, particularly, the physiological consequences of Hg bioaccumulation. In this study, we (i) describe the individual and temporal variation in blood concentration of total Hg (THg) over three breeding seasons, (ii) identify the factors that affect the THg exposure and (iii) determine the individual- and population-level connections between THg and blood-based markers of oxidative status in chicks of Scopoli's shearwaters (Calonectris diomedea) breeding on the island of Linosa in the southern Mediterranean. We carried out the work on chicks near fledging because they are fed with prey captured near the colony, thus their Hg levels reflect local contamination. The concentration of THg in erythrocytes varied from 0.23 to 4.29 µg g-1 dw. Chicks that were fed upon higher trophic level prey (i.e., higher δ15N values) had higher THg levels. Individual variation in THg concentrations was not explained by parental identity, sex nor δ13C values. There was significant variation in THg among chicks born from the same mother in different years. We found significant correlations between THg and markers of oxidative status; however, these correlations were no longer significant when we took into account the annual variation in mean values of all metrics. Males with higher values of body condition index had higher blood THg, while THg and body condition index were not correlated in females. Our data indicate that THg levels were moderate to high if compared to other seabirds. However, there is little evidence for harmful short-term detrimental effects owing to THg exposure.

Hippocampal-dependent familiar area map supports corrective re-orientation following navigational error during pigeon homing: a GPS-tracking study.

It is hypothesized that a central role of the vertebrate hippocampal formation (HF) in behavior is the learning and operation of a map-like representation of familiar landmarks and landscape features. One critical property of a map is that it should enable an individual to re-orient towards a goal location following a navigational error. To test this prediction on a spatial scale consistent with their naturally occurring behavior, control and HF-lesioned homing pigeons were trained from two locations and then subsequently released, while carrying portable GPS-tracking devices, following a phase-shift treatment. Analyses revealed that the HF-lesioned pigeons were less successful than control pigeons in re-orienting homewards following the phase-shift-induced error in their initial orientation. Furthermore, the observation that HF-lesioned pigeons were found to routinely ignore a land-sea landscape boundary when returning home from one of the release sites suggests that coarse landscape features may be an underappreciated source of navigational information for homing pigeons. The data demonstrate that, on a scale of tens of kilometers, homing pigeons are able to learn a hippocampal-dependent, map-like representation of familiar landmarks/landscape features that can support corrective re-orientation following a navigational error.

Waiting time before release increases the motivation to home in homing pigeons (Columba livia).

When performing homing experiments with individual releases, pigeons have to wait in a transport box for a certain amount of time before being released and hence perceive the departure of companions. Quite often, the last pigeons disappear straightforward from the release site. The question is whether this reflects improved orientation because of prolonged exposure to the release place or whether it reflects increased homing motivation. By releasing pigeons from a familiar site, we investigated the effects of the time spent at the release site on homing performance, recording pigeons' flights with GPS loggers. Our results show that, despite individual peculiarities of flight patterns, the waiting time at release site had a positive effect on homing speed and time, and reduced the time spent circling around the release point. However, the overall path efficiency as derived from GPS tracking was not influenced. These results suggest that a longer waiting time before release improves homing performance and this is related not only to increased navigational abilities but also to increased homing motivation.

Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles.

Transmission of prions between species is limited by the "species barrier," which hampers a full characterization of human prion strains in the mouse model. We report that the efficiency of primary transmission of prions from Creutzfeldt-Jakob disease patients to a wild rodent species, the bank vole (Clethrionomys glareolus), is comparable to that reported in transgenic mice carrying human prion protein, in spite of a low prion protein-sequence homology between man and vole. Voles infected with sporadic and genetic Creutzfeldt-Jakob disease isolates show strain-specific patterns of spongiform degeneration and pathological prion protein-deposition, and accumulate protease-resistant prion protein with biochemical properties similar to the human counterpart. Adaptation of genetic Creutzfeldt-Jakob disease isolates to voles shows little or no evidence of a transmission barrier, in contrast to the striking barriers observed during transmission of mouse, hamster, and sheep prions to voles. Our results imply that in voles there is no clear relationship between the degree of homology of the prion protein of the donor and recipient species and susceptibility, consistent with the view that the prion strain gives a major contribution to the species barrier. The vole is therefore a valuable model to study human prion diversity and, being susceptible to a range of animal prions, represents a unique tool for comparing isolates from different species.

PCBs in the eggs of Eurasian kestrels indicate exposure to local pollution.

Fail-to-hatch kestrel (Falco tinnunculus) eggs collected at the end of the 1999 and 2005 breeding seasons from nest boxes in and around the city of Rome, Italy, were analyzed by gas chromatography with electron capture detection for their PCB content and for the presence of DDT derivatives and other organochlorines. Among the various PCBs, congeners 153 and 180 were detected in all the eggs and showed the highest concentrations. Eggs collected from the same nest from a polluted location in Rome during 2 different years showed similar type and number of PCB congeners. These data and the fact that eggs from another nest near a sulphate mine had, atypically, low-chlorinated congeners support the conclusion that eggs of this species, whose adults in the Mediterranean and continental Europe perform only short or no migration movements, might be indicative of local pollution. When multiple eggs in the same clutch were analyzed, the PCBs were similar in type but their concentration decreased within clutch, likely in parallel to the laying order.

A new use of technology to solve an old problem: Estimating the population size of a burrow nesting seabird.

Estimating the population of burrow-nesting seabirds is a challenging task, as human presence in the colony creates disturbances and can damage burrows and occupants. Here, we present a novel method using aerial photographs taken with Unmanned Aerial Vehicles (UAVs) to estimate the population size of a burrow-nesting seabird, the Black-vented Shearwater (Puffinus opisthomelas), on Natividad Island, Mexico. Our results provide a census of burrows in the colony, with very low detection error (5.6%). This is greater accuracy compared to other methods based on extrapolating results from sample plots to total colony area. We then combined this burrow census with ground truth data on occupancy to estimate population size. We obtained a population estimate of 37,858 and 46,322 breeding pairs for 2016 and 2017 respectively. The proposed method provides a cost effective and repeatable approach for monitoring numbers of burrows occupied in a colony, thereby enabling easier and faster estimates of population trends. We suggest this method can be valid for other burrow-nesting species in habitats without dense vegetation cover.

Pigeon homing along highways and exits.

BACKGROUND: Anecdotal observations and early airplane and helicopter tracking studies suggest that pigeons sometimes follow large roads and use landmarks as turning points during their homeward journey. However, technical limitations in tracking pigeon routes have prevented proof. RESULTS: Here, we present experimental and statistical evidence for this strategy from the analysis of 216 GPS-recorded pigeon tracks over distances up to 50 km. Experienced pigeons released from familiar sites during 3 years around Rome, Italy, were significantly attracted to highways and a railway track running toward home, in many cases without anything forcing them to follow such guide-rails. Birds often broke off from the highways when these veered away from home, but many continued their flight along the highway until a major junction, even when the detour added substantially to their journey. The degree of road following increased with repeated releases but not flight length. Significant road following (in 40%-50% of the tracks) was mainly observed from release sites along northwest-southeast axis. CONCLUSIONS: Our data demonstrate the existence of a learned road-following homing strategy of pigeons and the use of particular topographical points for final navigation to the loft. Apparently, the better-directed early stages of the flight compensated the added final detour. During early and middle stages of the flight, following large and distinct roads is likely to reflect stabilization of a compass course rather than the presence of a mental roadmap. A cognitive (roadmap) component manifested by repeated crossing of preferred topographical points, including highway exits, is more likely when pigeons approach the loft area. However, it might only be expected in pigeons raised in an area characterized by navigationally relevant highway systems.

Combined use of tri-axial accelerometers and GPS reveals the flexible foraging strategy of a bird in relation to weather conditions.

Tri-axial accelerometry has proved to be a useful technique to study animal behavior with little direct observation, and also an effective way to measure energy expenditure, allowing a refreshing revisit to optimal foraging theory. This theory predicts that individuals should gain the most energy for the lowest cost in terms of time and energy when foraging, in order to maximize their fitness. However, during a foraging trip, central-place foragers could face different trade-offs during the commuting and searching parts of the trip, influencing behavioral decisions. Using the lesser kestrel (Falco naumanni) as an example we study the time and energy costs of different behaviors during the commuting and searching parts of a foraging trip. Lesser kestrels are small insectivorous falcons that behave as central-place foragers during the breeding season. They can commute by adopting either time-saving flapping flights or energy-saving soaring-gliding flights, and capture prey by using either time-saving active hovering flights or energy-saving perch-hunting. We tracked 6 lesser kestrels using GPS and tri-axial accelerometers during the breeding season. Our results indicate that males devoted more time and energy to flight behaviors than females in agreement with being the sex responsible for food provisioning to the nest. During the commuting flights, kestrels replaced flapping with soaring-gliding flights as solar radiation increased and thermal updrafts got stronger. In the searching part, they replaced perch-hunting with hovering as wind speed increased and they experienced a stronger lift. But also, they increased the use of hovering as air temperature increased, which has a positive influence on the activity level of the preferred prey (large grasshoppers). Kestrels maintained a constant energy expenditure per foraging trip, although flight and hunting strategies changed dramatically with weather conditions, suggesting a fixed energy budget per trip to which they adjusted their commuting and searching strategies in response to weather conditions.