The great tit HapMap project: A continental-scale analysis of genomic variation in a songbird.

A major aim of evolutionary biology is to understand why patterns of genomic diversity vary within taxa and space. Large-scale genomic studies of widespread species are useful for studying how environment and demography shape patterns of genomic divergence. Here, we describe one of the most geographically comprehensive surveys of genomic variation in a wild vertebrate to date; the great tit (Parus major) HapMap project. We screened ca 500,000 SNP markers across 647 individuals from 29 populations, spanning ~30 degrees of latitude and 40 degrees of longitude - almost the entire geographical range of the European subspecies. Genome-wide variation was consistent with a recent colonisation across Europe from a South-East European refugium, with bottlenecks and reduced genetic diversity in island populations. Differentiation across the genome was highly heterogeneous, with clear 'islands of differentiation', even among populations with very low levels of genome-wide differentiation. Low local recombination rates were a strong predictor of high local genomic differentiation (FST), especially in island and peripheral mainland populations, suggesting that the interplay between genetic drift and recombination causes highly heterogeneous differentiation landscapes. We also detected genomic outlier regions that were confined to one or more peripheral great tit populations, probably as a result of recent directional selection at the species' range edges. Haplotype-based measures of selection were related to recombination rate, albeit less strongly, and highlighted population-specific sweeps that likely resulted from positive selection. Our study highlights how comprehensive screens of genomic variation in wild organisms can provide unique insights into spatio-temporal evolutionary dynamics.

Expression patterns of heat-shock genes during stopover and the trade-off between refueling and stress response in a passerine migrant.

Migrating birds are often exposed to variable environments and face a multitude of stress exposures along their long-distance flights. During stopover refueling, migratory birds must balance the need to accumulate energy reserves to continue their migration with the need to respond to environmental and physiological stressors. We examined the gene expression patterns of different Heat Shock Proteins (HSPs) in migrating birds during stopover at different body condition states (lean vs. fat), to provide some first insights on the role of HSPs in bird migration and explore the concept of a trade-off between refueling and stress response. Our results showed upregulation of HSP expression at release that could be associated with muscle growth and increased cholesterol and lipid synthesis needed for birds to fuel their upcoming migration. On the other hand, during capture, upregulation of HSP5 could be attributed to physiological recovery from the non-stop endurance flight when crossing the Sahara Desert-Mediterranean Sea ecological barrier. All birds significantly increased their fuel loads up to 48% of lean body mass and we provide evidence for muscle rebuilding during stopover as flight muscle mass increased by 10%, highlighting the fact that stopover sites can play a major role in the physiological recovery of migrants.

Adaptive Regulation of Stopover Refueling during Bird Migration: Insights from Whole Blood Transcriptomics.

Migration is one of the most energy-demanding tasks in avian life cycle. Many birds might not have sufficient fuel stores to cover long distances, so they must stop to rest and refuel at stopover sites, especially after the crossing of large ecological barriers. There, birds undergo several behavioral, morphological, and physiological trait adjustments to recover from and prepare for their journey; however, regulation of such processes at the molecular level remains largely unknown. In this study, we used transcriptomic information from the whole blood of migrating garden warblers (Sylvia borin) to identify key regulatory pathways related to adaptations for migration. Birds were temporarily caged during spring migration stopover and then sampled twice at different refueling states (lean vs. fat), reflecting different migratory stages (stopover arrival vs. departure) after the crossing of an extended ecological barrier. Our results show that top expressed genes during migration are involved in important pathways regarding adaptations to migration at high altitudes such as increase of aerobic capacity and angiogenesis. Gene expression profiles largely reflected the two experimental conditions with several enzymes involved in different aspects of metabolic activity being differentially expressed between states providing several candidate genes for future functional studies. Additionally, we identified several hub genes, upregulated in lean birds that could be involved in the extraordinary phenotypic flexibility in organ mass displayed by avian migrants. Finally, our approach provides novel evidence that regulation of water homeostasis may represent a significant adaptive mechanism, allowing birds to conserve water during long-distance flight, mainly through protein catabolism.

Acoustic Monitoring Confirms Significant Poaching Pressure of European Turtle Doves (Streptopelia turtur) during Spring Migration across the Ionian Islands, Greece.

The European turtle dove (Streptopelia turtur) is an Afro-Palearctic migrant whose populations have declined by 79% from 1980 to 2014. In 2018, the International Single Species Action Plan for the Turtle Dove (ISSAP) was developed with the goal of enabling, by 2028, an increase in turtle dove numbers along each of the three migration flyways (western, central, eastern). To achieve this, the illegal killing of turtle doves, a critical threat to the species, has to be eradicated. The Ionian Islands off the west coast of Greece lie on the eastern flyway and are considered a major turtle dove poaching hot-spot during spring migration. Quantifying wildlife crime, however, is challenging. In the absence of a reliable protocol for monitoring spring poaching levels, the agencies tasked with tackling the problem have no means of assessing the effectiveness of the anti-poaching measures and adapting them if required. Using passive acoustic monitoring (PAM) methods, we recorded gun hunting intensity at known turtle dove poaching sites during the 2019-2022 spring migrations (2-10 sites/season) with unprecedented spatial and temporal resolution. Based on published gunshot to killed/injured bird ratio for similar species (corroborated with discussions with local hunters) and an estimate of the proportion of hunting sites monitored by our PAM grid (using gunshot detection range estimates from control gunshots), we estimated that in 2021, up to 57,095 turtle doves were killed or injured across five Ionian Islands (Zakynthos, Paxi, Antipaxi, Othoni, and Mathraki). The 2022 estimate was almost half, but it is unclear as to whether the change is due to a decline in poachers or turtle doves. We propose ways of improving confidence in future estimates, and call for a temporary moratorium of autumn turtle dove hunting in Greece-as per ISSAP recommendation-until spring poaching is eradicated and the eastern flyway population shows signs of a full recovery. Finally, we hope our findings will pave the way for the development of PAM grids at turtle dove poaching hot-spots across all migration flyways, contributing to the global conservation of the species.

Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes.

The migratory behavior of wild birds contributes to the geographical spread of ticks and their microorganisms. In this study, we aimed to investigate the dispersal and co-occurrence of Francisella and spotted fever group Rickettsia (SFGR) in ticks infesting birds migrating northward in the African-Western Palaearctic region (AWPR). Birds were trapped with mist nests across the Mediterranean basin during the 2014 and 2015 spring migration. In total, 575 ticks were collected from 244 birds. We screened the ticks for the species Francisella tularensis, the genus Francisella, and SFGR by microfluidic real-time PCR. Confirmatory analyses and metagenomic sequencing were performed on tick samples that putatively tested positive for F. tularensis during initial screenings. Hyalomma rufipes was the most common tick species and had a high prevalence of Francisella, including co-occurrence of Francisella and SFGR. Metagenomic analysis of total DNA extracted from two H. rufipes confirmed the presence of Francisella, Rickettsia, and Midichloria. Average nucleotide identity and phylogenetic inference indicated the highest identity of the metagenome-assembled genomes to a Francisella-like endosymbiont (FLE), Rickettsia aeschlimannii, and Midichloria mitochondrii. The results of this study suggest that (i) FLE- and SFGR-containing ticks are dispersed by northbound migratory birds in the AWPR, (ii) H. rufipes likely is not involved in transmission of F. tularensis in the AWPR, and (iii) a dual endosymbiosis of FLEs and Midichloria may support some of the nutritional requirements of H. rufipes.

Association between guilds of birds in the African-Western Palaearctic region and the tick species Hyalomma rufipes, one of the main vectors of Crimean-Congo hemorrhagic fever virus.

INTRODUCTION: The ecology of the vertebrate host contributes to the geographical range expansion of ticks. In this study, we investigated which tick taxa that infest and are dispersed by birds along African-Western Palaearctic flyways during northward migration, and whether bird ecology was associated with tick taxa. MATERIALS AND METHODS: Ticks were collected from birds trapped at bird observatories in Spain, Italy, Greece, and Israel during the spring migration of 2014 and 2015, using mist nets. The tick-infested bird species were classified into guilds, using different combinations of the variables: migration distance, wintering region, foraging behaviour, and winter habitat. Ticks were molecularly determined to genus and species level by sequencing fragments of the 12S ribosomal DNA (rDNA) gene and by phylogenetic inference, using the Maximum Likelihood algorithm. Data were analysed using descriptive measures, graphs, Chi2 tests, the Tukey-Kramer test, and a parametric linear model (generalized linear model) in order to analyse and adjust for characteristics in the bird guilds and their relationship to collected tick taxa. RESULTS: Most (84.2%) of the 10,209 trapped birds were long-distance migrants, of which 2.4% were infested by ticks. The most common tick species was Hyalomma rufipes (77.7%; 447/575), a known vector and reservoir of Crimean-Congo hemorrhagic fever virus. Bird guilds containing only long-distance migrants with wintering areas in Africa were associated with the tick species H. rufipes (p < 0.0001). Furthermore, bird winter habitat was associated with H. rufipes (p = 0.003); with bird species overwintering in open habitat (p = 0.014) and wetlands (p = 0.046) having significantly more H. rufipes as compared to birds with a winter habitat comprising forest and shrubs (p = 0.82). CONCLUSIONS: With climate change, the likelihood of establishment of permanent Hyalomma populations in central and northern Europe is increasing. Thus, surveillance programs for monitoring the risk of introduction and establishment of H. rufipes in the Western-Palaearctic should be established. Our study suggests that migratory bird species wintering in African open habitats and wetlands are good candidates for monitoring potential introduction.

Prevalence and genetic diversity of avian haemosporidian parasites in wild bird species of the order Columbiformes.

Diseases can play a role in species decline. Among them, haemosporidian parasites, vector-transmitted protozoan parasites, are known to constitute a risk for different avian species. However, the magnitude of haemosporidian infection in wild columbiform birds, including strongly decreasing European turtle doves, is largely unknown. We examined the prevalence and diversity of haemosporidian parasites Plasmodium, Leucocytozoon and subgenera Haemoproteus and Parahaemoproteus in six species of the order Columbiformes during breeding season and migration by applying nested PCR, one-step multiplex PCR assay and microscopy. We detected infections in 109 of the 259 screened individuals (42%), including 15 distinct haemosporidian mitochondrial cytochrome b lineages, representing five H. (Haemoproteus), two H. (Parahaemoproteus), five Leucocytozoon and three Plasmodium lineages. Five of these lineages have never been described before. We discriminated between single and mixed infections and determined host species-specific prevalence for each parasite genus. Observed differences among sampled host species are discussed with reference to behavioural characteristics, including nesting and migration strategy. Our results support previous suggestions that migratory birds have a higher prevalence and diversity of blood parasites than resident or short-distance migratory species. A phylogenetic reconstruction provided evidence for H. (Haemoproteus) as well as H. (Parahaemoproteus) infections in columbiform birds. Based on microscopic examination, we quantified parasitemia, indicating the probability of negative effects on the host. This study provides a large-scale baseline description of haemosporidian infections of wild birds belonging to the order Columbiformes sampled in the northern hemisphere. The results enable the monitoring of future changes in parasite transmission areas, distribution and diversity associated with global change, posing a potential risk for declining avian species as the European turtle dove.

Spatially different annual cycles but similar haemosporidian infections in distant populations of collared sand martins.

BACKGROUND: Populations of long-distance migratory birds experience different environments and are consequently exposed to different parasites throughout their annual cycles. Though, specific whereabouts and accompanied host-parasite interactions remain unknown for most migratory passerines. Collared sand martins (Riparia riparia) breeding in the western Palaearctic spend the nonbreeding period in Africa, but it is not yet clear whether specific populations differ in overwintering locations and whether these also result in varying infections with vector-transmitted endoparasites. RESULTS: Geolocator tracking revealed that collared sand martins from northern-central and central-eastern Europe migrate to distant nonbreeding sites in West Africa and the Lake Chad basin in central Africa, respectively. While the ranges of these populations were clearly separated throughout the year, they consistently spent up to 60% of the annual cycle in Africa. Ambient light recorded by geolocators further indicated unsheltered roosting during the nonbreeding season in Africa compared to the breeding season in Europe. We found 5-26% prevalence of haemosporidian parasites in three breeding populations and one migratory passage population that was only sampled but not tracked. In total, we identified seven Plasmodium and nine Haemoproteus lineages (incl. two and seven new lineages, respectively), the latter presumably typical for swallows (Hirundinae) hosts. 99.5% of infections had a low intensity, typical for chronic infection stages, whereas three individuals (0.5%) showed high parasitaemia typical for acute infections during spring migration and breeding. CONCLUSIONS: Our study shows that blood parasite infections are common in several western Palaearctic breeding populations of collared sand martins who spent the nonbreeding season in West Africa and the lake Chad region. Due to long residency at the nonbreeding grounds blood parasite transmissions may mainly occur at host population-specific residences sites in Europe and Africa; the latter being likely facilitated by unsheltered roosting and thus high vulnerability to hematophagous insects. The rare cases of high parasitaemia during spring migration and breeding further indicates either relapses of chronic infection or primary infections which occurred shortly before migration and during breeding.

Mining citizen science data to explore stopover sites and spatiotemporal variation in migration patterns of the red-footed falcon.

Citizen science data have already been used to effectively address questions regarding migration, a fundamental stage in the life history of birds. In this study, we use data from eBird and from 3 additional regional citizen science databases to describe the migration routes and timing of the red-footed falcon Falco vespertinus in the Mediterranean region across 8 years (2010-2017). We further examine the seasonal and yearly variation in migration patterns and explore sites used during the species migration. Our results suggest that the autumn passage is spatially less variable and temporally more consistent among years than in spring and that birds migrate faster in spring than in autumn. The species seems to be more prevalent along the Central Mediterranean during spring migration, probably as a result of the clockwise loop migration that red-footed falcons perform. There was a high variation in annual median migration dates for both seasons as well as in migration routes across years and seasons. Higher variation was exhibited in the longitudinal component thus indicating flexibility in migration routes. In addition, our results showed the species' preference for lowlands covered with cropland and mosaics of cropland and natural vegetation as stopover sites during migration. Stopover areas predicted from our distribution modeling highlight the importance of the Mediterranean islands as stopover sites for sea-crossing raptors, such as the red-footed falcon. This study is the first to provide a broad-scale spatiotemporal perspective on the species migration across seasons, years and flyways and demonstrates how citizen science data can inform future monitoring and conservation strategies.

A divergent Anaplasma phagocytophilum variant in an Ixodes tick from a migratory bird; Mediterranean basin.

Anaplasma phagocytophilum (AP) has vast geographical and host ranges and causes disease in humans and domesticated animals. We investigated the role of northward migratory birds in the dispersal of tick-borne AP in the African-Western Palearctic. Ticks were collected from northward migratory birds trapped during spring migration of 2010 at two localities in the central Mediterranean Sea. AP DNA was detected by PCR (gltA and 16S rRNA) and variant determination was performed using ankA sequences. In total, 358 ticks were collected. One of 19 ticks determined as Ixodes was confirmed positive for AP DNA. The tick was collected from a woodchat shrike (Lanius senator senator) trapped in Greece, and molecularly determined to belong to the I. ricinus complex and sharing highest (95%) 16S RNA sequence identity to I. gibbosus. The ankA AP sequence exhibited highest similarity to sequences from rodents and shrews (82%) and ruminants (80%). Phylogenetic analyses placed it convincingly outside other clades, suggesting that it represents a novel AP variant. The divergent Ixodes species harboring a novel AP variant could either indicate an enzootic cycle involving co-evolution with birds, or dissemination from other regions by avian migration. None of the 331 Hyalomma marginatum sensu lato ticks, all immature stages, were positive for AP DNA, lending no evidence for the involvement of Hyalomma ticks transported by birds in the ecology of AP.

Assessing the seasonal and intrinsic variability of neurotoxic and cyto-genotoxic biomarkers in blood of free-living Eleonoras' falcons.

In the present study we investigated seasonal and intrinsic variability of neurotoxic and cyto-genotoxic biomarkers in blood plasma and erythrocytes of free-living Eleonoras' falcons, captured during the pre-breeding (May of 2017 and 2018) and breeding period (September of 2017) on the Antikythira Island (Greece). Specifically, blood samples of captured birds were prepared for the determination of cholinesterase (ChEs, i.e. acetylcholinesterase/AChE and butyrylcholinesterase/ BChE) activity, as well as the formation of nuclear (i.e. the formation of micronuclei into the cells/MN, binucleated cells/BN and others), and cellular/cytoplasmic (i.e. echinocytes/EC, acanthocytes/AC and notched cells/NC) abnormalities in blood plasma and erythrocytes, respectively. Our results indicated that birds sampled in late May had higher ChE and BChE activity levels, as well as higher frequency of total nuclear abnormalities. The latter were also higher in second calendar year (2cy) birds. Cellular/cytoplasmic abnormalities were less frequent in falcons having better body condition, sampled in late May, as well as in light-morph falcons. The observed ChEs activities, as well as nuclear and cellular/cytoplasmic abnormalities revealed that Eleonora's falcons are likely to be exposed to chemical agents with neurotoxic and cyto-genotoxic potential year round, while different aspects of their biology and ecology, such as their reproductive and nutritional status, could mediate their levels. Although we encourage more sampling campaigns to verify the identified seasonal and intrinsic sources of variation in biomarkers tested, the current study enriches the existing knowledge about their usefulness in the environmental monitoring and risk assessment of migratory birds, like Eleonoras' falcon.

Alkhurma Hemorrhagic Fever Virus RNA in Hyalomma rufipes Ticks Infesting Migratory Birds, Europe and Asia Minor.

Alkhurma hemorrhagic fever virus RNA was detected in immature Hyalomma rufipes ticks infesting northward migratory birds caught in the North Mediterranean Basin. This finding suggests a role for birds in the ecology of the Alkhurma hemorrhagic fever virus and a potential mechanism for dissemination to novel regions. Increased surveillance is warranted.

Habitat selection of woodchat shrikes Lanius senator during spring stopover is related to foraging strategy.

Every spring a huge number of passerines cross the Sahara Desert and the Mediterranean Sea on their way to their breeding grounds. Stopover sites after such extended barriers where birds can rest, refuel, and find shelter from adverse weather, are of crucial importance for the outcome of their migration. Stopover habitat selection used by migrating birds depends on landscape context, habitat patch characteristics, as well as on the particular energetic conditions and needs of individual birds, but it is still poorly investigated. We focused on a long-distance migrating passerine, the woodchat shrike, in order to investigate for the first time the species' habitat selection at a spring stopover site (island of Antikythira, Greece) after the crossing of the Sahara Desert and Mediterranean Sea. We implemented radio-tracking, color-ringing, and visual behavioral observations to collect data on microhabitat use. Generalized Linear Mixed Models were developed to identify the species' most preferred microhabitat during its stopover on this low human disturbed island. We found that high maquis vegetation surrounded by low vegetation was chosen as perches for hunting. Moreover, high maquis vegetation appeared to facilitate hunting attempts toward the ground, the most frequently observed foraging strategy. Finally, we discuss our findings in the context of conservation practices for the woodchat shrike and their stopover sites on Mediterranean islands.

Spotted fever Rickettsia species in Hyalomma and Ixodes ticks infesting migratory birds in the European Mediterranean area.

BACKGROUND: A few billion birds migrate annually between their breeding grounds in Europe and their wintering grounds in Africa. Many bird species are tick-infested, and as a result of their innate migratory behavior, they contribute significantly to the geographic distribution of pathogens, including spotted fever rickettsiae. The aim of the present study was to characterize, in samples from two consecutive years, the potential role of migrant birds captured in Europe as disseminators of Rickettsia-infected ticks. METHODS: Ticks were collected from a total of 14,789 birds during their seasonal migration northwards in spring 2009 and 2010 at bird observatories on two Mediterranean islands: Capri and Antikythira. All ticks were subjected to RNA extraction followed by cDNA synthesis and individually assayed with a real-time PCR targeting the citrate synthase (gltA) gene. For species identification of Rickettsia, multiple genes were sequenced. RESULTS: Three hundred and ninety-eight (2.7%) of all captured birds were tick-infested; some birds carried more than one tick. A total number of 734 ticks were analysed of which 353 ± 1 (48%) were Rickettsia-positive; 96% were infected with Rickettsia aeschlimannii and 4% with Rickettsia africae or unidentified Rickettsia species. The predominant tick taxon, Hyalomma marginatum sensu lato constituted 90% (n = 658) of the ticks collected. The remaining ticks were Ixodes frontalis, Amblyomma sp., Haemaphysalis sp., Rhipicephalus sp. and unidentified ixodids. Most ticks were nymphs (66%) followed by larvae (27%) and adult female ticks (0.5%). The majority (65%) of ticks was engorged and nearly all ticks contained visible blood. CONCLUSIONS: Migratory birds appear to have a great impact on the dissemination of Rickettsia-infected ticks, some of which may originate from distant locations. The potential ecological, medical and veterinary implications of such Rickettsia infections need further examination.

Fuelling in front of the barrier-are there age based behavioral differences in Garden Warblers Sylvia borin?

Garden Warblers Sylvia borin were studied during autumn stopover in Crete before crossing the barrier of the Mediterranean Sea and the Sahara Desert. Birds followed with transmitters show extensive stopover periods, which were longer in first-year birds, 16 days, compared with adult birds, 14 days. The distribution of body masses from birds trapped in fig trees were used to estimate the departure body mass and the results found indicate that both age categories on average depart with a fuel load close to 100% of lean body mass. The movement of transmitter birds shows differences between first-year and adult birds. Adult birds move further away from the release site and many also left the study area. Several were found settled outside the study area, up to 17 km away, indicating that they regularly make longer stopover movements. It is suggested that this might be a result of that they return to a place where they stayed during an earlier migration. It was shown that stopover site fidelity exists and nine garden warblers were recaptured in the area during a following autumn. The results found highlights the importance of stopover areas close to the Sahara Desert.

On the potential roles of ticks and migrating birds in the ecology of West Nile virus.

BACKGROUND: Mosquitoes are the primary vectors of West Nile virus (WNV). Ticks have, however, been suggested to be potential reservoirs of WNV. In order to investigate their role in the spread of the virus, ticks, which had been collected from birds migrating northwards from Africa to Europe, were analyzed for the potential presence of WNV-RNA. METHODS: On the Mediterranean islands Capri and Antikythira a total of 14,824 birds were captured and investigated from which 747 ticks were collected. RESULTS AND CONCLUSION: Most of the identified ticks (93%) were nymphs and larvae of Hyalomma marginatum sensu lato, most of which were or appear to be Hyalomma rufipes. Of these ticks 729 were individually screened for WNV-RNA. None of the ticks was found to be WNV positive. Thus, there was no evidence that Hyalomma marginatum s.l. ticks play a role in the spread of WNV from Africa to Europe.

The eyes have it: long-distance dispersal by an intraorbital leech parasite of birds.

A leech was found parasitizing the ocular orbit of a common redstart captured during a faunistic survey of Antikythira in the Aegean Sea during the spring migration of 2012. Morphological and molecular characterizations placed the leech in the mucous-membrane specific leech family Praobdellidae and definitively as the species Parapraobdella lineata. This is the first record of any leech parasitizing a passerine bird, Phoenicurus phoenicurus , and the first of a praobdellid leech on any avian host.