Hibernating vesper bats are a weak source for biomonitoring of coronaviruses.

Background: Our study explores the role of bats as reservoirs of coronaviruses. Methods: We conducted virological screening of bats hibernating in military bunkers at the Natura 2000 site "Nietoperek" in Western Poland collecting oral and anal swab samples from 138 bats across six species to apply a combination of pan-coronavirus and SARS-CoV-2 specific PCR assays. Results: Only one anal swab tested positive for coronavirus. No SARS-CoV-2 was detected in any of the samples. The low prevalence of coronavirus in the studied colony contrasts with higher rates found in other regions and may be influenced by hibernation. Conclusions: Hibernating bats may show a low prevalence of coronavirus, potentially due to the hibernation process itself. This finding indicates that hibernating bats may not be the most optimal subjects for screening zoonotic pathogens. However, biomonitoring of bats for emerging and reemerging diseases is recommended for comprehensive epidemiological insights.

Monitoring mosquito richness in an understudied area: can environmental DNA metabarcoding be a complementary approach to adult trapping?

Mosquito surveillance programmes are essential to assess the risks of local vector-borne disease outbreaks as well as for early detection of mosquito invasion events. Surveys are usually performed with traditional sampling tools (i.e., ovitraps and dipping method for immature stages or light or decoy traps for adults). Over the past decade, numerous studies have highlighted that environmental DNA (eDNA) sampling can enhance invertebrate species detection and provide community composition metrics. However, the usefulness of eDNA for detection of mosquito species has, to date, been largely neglected. Here, we sampled water from potential larval breeding sites along a gradient of anthropogenic perturbations, from the core of an oil palm plantation to the rainforest on São Tomé Island (Gulf of Guinea, Africa). We showed that (i) species of mosquitoes could be detected via metabarcoding mostly when larvae were visible, (ii) larvae species richness was greater using eDNA than visual identification and (iii) new mosquito species were also detected by the eDNA approach. We provide a critical discussion of the pros and cons of eDNA metabarcoding for monitoring mosquito species diversity and recommendations for future research directions that could facilitate the adoption of eDNA as a tool for assessing insect vector communities.

Chromosome-level assembly of the Rangifer tarandus genome and validation of cervid and bovid evolution insights.

BACKGROUND: Genome assembly into chromosomes facilitates several analyses including cytogenetics, genomics and phylogenetics. Despite rapid development in bioinformatics, however, assembly beyond scaffolds remains challenging, especially in species without closely related well-assembled and available reference genomes. So far, four draft genomes of Rangifer tarandus (caribou or reindeer, a circumpolar distributed cervid species) have been published, but none with chromosome-level assembly. This emblematic northern species is of high interest in ecological studies and conservation since most populations are declining. RESULTS: We have designed specific probes based on Oligopaint FISH technology to upgrade the latest published reindeer and caribou chromosome-level genomes. Using this oligonucleotide-based method, we found six mis-assembled scaffolds and physically mapped 68 of the largest scaffolds representing 78% of the most recent R. tarandus genome assembly. Combining physical mapping and comparative genomics, it was possible to document chromosomal evolution among Cervidae and closely related bovids. CONCLUSIONS: Our results provide validation for the current chromosome-level genome assembly as well as resources to use chromosome banding in studies of Rangifer tarandus.

Serologic Surveillance for SARS-CoV-2 Infection among Wild Rodents, Europe.

We report results from serologic surveillance for exposure to SARS-CoV-2 among 1,237 wild rodents and small mammals across Europe. All samples were negative, with the possible exception of 1. Despite suspected potential for human-to-rodent spillover, no evidence of widespread SARS-CoV-2 circulation in rodent populations has been reported to date.Esitämme tulokset serologisesta tutkimuksesta, jossa seulottiin SARS-CoV-2 tartuntojen varalta 1,237 luonnonvaraista jyrsijää ja piennisäkästä eri puolilta Eurooppaa. Kaikki näytteet olivat negatiivisia, yhtä näytettä lukuun ottamatta. SARS-CoV-2:n läikkymisen ihmisistä jyrsijöihin on arveltu olevan mahdollista, mutta todisteet viruksen laajamittaisesta leviämisestä jyrsijäpopulaatioissa puuttuvat.

Design and validation of a 63K genome-wide SNP-genotyping platform for caribou/reindeer (Rangifer tarandus).

BACKGROUND: Development of large single nucleotide polymorphism (SNP) arrays can make genomic data promptly available for conservation problematic. Medium and high-density panels can be designed with sufficient coverage to offer a genome-wide perspective and the generated genotypes can be used to assess different genetic metrics related to population structure, relatedness, or inbreeding. SNP genotyping could also permit sexing samples with unknown associated metadata as it is often the case when using non-invasive sampling methods favored for endangered species. Genome sequencing of wild species provides the necessary information to design such SNP arrays. We report here the development of a SNP-array for endangered Rangifer tarandus using a multi-platform sequencing approach from animals found in diverse populations representing the entire circumpolar distribution of the species. RESULTS: From a very large comprehensive catalog of SNPs detected over the entire sample set (N = 894), a total of 63,336 SNPs were selected. SNP selection accounted for SNPs evenly distributed across the entire genome (~ every 50Kb) with known minor alleles across populations world-wide. In addition, a subset of SNPs was selected to represent rare and local alleles found in Eastern Canada which could be used for ecotype and population assignments - information urgently needed for conservation planning. In addition, heterozygosity from SNPs located in the X-chromosome and genotyping call-rate of SNPs located into the SRY gene of the Y-chromosome yielded an accurate and robust sexing assessment. All SNPs were validated using a high-throughput SNP-genotyping chip. CONCLUSION: This design is now integrated into the first genome-wide commercially available genotyping platform for Rangifer tarandus. This platform would pave the way to future genomic investigation of populations for this endangered species, including estimation of genetic diversity parameters, population assignments, as well as animal sexing from genetic SNP data for non-invasive samples.

Wild Red Deer (Cervus elaphus) Do Not Play a Role as Vectors or Reservoirs of SARS-CoV-2 in North-Eastern Poland.

Several studies reported a high prevalence of SARS-CoV-2 among white-tailed deer in North America. Monitoring cervids in all regions to better understand SARS-CoV-2 infection and circulation in other deer populations has been urged. To evaluate deer exposure and/or infection to/by SARS-CoV-2 in Poland, we sampled 90 red deer shot by hunters in five hunting districts in north-eastern Poland. Serum and nasopharyngeal swabs were collected, and then an immunofluorescent assay (IFA) to detect anti-SARS-CoV-2 antibodies was performed as well as real-time PCR with reverse transcription for direct virus detection. No positive samples were detected. There is no evidence of spillover of SARS-CoV-2 from the human to deer population in Poland.

CNVs with adaptive potential in Rangifer tarandus: genome architecture and new annotated assembly.

Rangifer tarandus has experienced recent drastic population size reductions throughout its circumpolar distribution and preserving the species implies genetic diversity conservation. To facilitate genomic studies of the species populations, we improved the genome assembly by combining long read and linked read and obtained a new highly accurate and contiguous genome assembly made of 13,994 scaffolds (L90 = 131 scaffolds). Using de novo transcriptome assembly of RNA-sequencing reads and similarity with annotated human gene sequences, 17,394 robust gene models were identified. As copy number variations (CNVs) likely play a role in adaptation, we additionally investigated these variations among 20 genomes representing three caribou ecotypes (migratory, boreal and mountain). A total of 1,698 large CNVs (length > 1 kb) showing a genome distribution including hotspots were identified. 43 large CNVs were particularly distinctive of the migratory and sedentary ecotypes and included genes annotated for functions likely related to the expected adaptations. This work includes the first publicly available annotation of the caribou genome and the first assembly allowing genome architecture analyses, including the likely adaptive CNVs reported here.

Metabarcoding options to study eukaryotic endoparasites of birds.

There is growing interest in the study of avian endoparasite communities, and metabarcoding is a promising approach to complement more conventional or targeted methods. In the case of eukaryotic endoparasites, phylogenetic diversity is extreme, with parasites from 4 kingdoms and 11 phyla documented in birds. We addressed this challenge by comparing different primer sets across 16 samples from 5 bird species. Samples consisted of blood, feces, and controlled mixes with known proportions of bird and nematode DNA. Illumina sequencing revealed that a 28S primer set used in combination with a custom blocking primer allowed detection of various plasmodiid parasites and filarioid nematodes in the blood, coccidia in the feces, as well as two potentially pathogenic fungal groups. When tested on the controlled DNA mixes, these primers also increased the proportion of nematode DNA by over an order of magnitude. An 18S primer set, originally designed to exclude metazoan sequences, was the most effective at reducing the relative number of avian DNA sequences and was the only one to detect Trypanosoma in the blood. Expectedly, however, it did not allow nematode detection and also failed to detect avian malaria parasites. This study shows that a 28S set including a blocking primer allows detection of several major and very diverse bird parasite clades, while reliable amplification of all major parasite groups may require a combination of markers. It helps clarify options for bird parasite metabarcoding, according to priorities in terms of the endoparasite clades and the ecological questions researchers wish to focus on.

Past, present and future contributions of evolutionary biology to wildlife forensics, management and conservation.

Successfully implementing fundamental concepts into concrete applications is challenging in any given field. It requires communication, collaboration and shared will between researchers and practitioners. We argue that evolutionary biology, through research work linked to conservation, management and forensics, had a significant impact on wildlife agencies and department practices, where new frameworks and applications have been implemented over the last decades. The Quebec government's Wildlife Department (MFFP: Ministère des Forêts, de la Faune et des Parcs) has been proactive in reducing the "research-implementation" gap, thanks to prolific collaborations with many academic researchers. Among these associations, our department's outstanding partnership with Dr. Louis Bernatchez yielded significant contributions to harvest management, stocking programmes, definition of conservation units, recovery of threatened species, management of invasive species and forensic applications. We discuss key evolutionary biology concepts and resulting concrete examples of their successful implementation that derives directly or indirectly from this successful partnership. While old and new threats to wildlife are bringing new challenges, we expect recent developments in eDNA and genomics to provide innovative solutions as long as the research-implementation bridge remains open.

Impact of Annual Bacterial Epizootics on Albatross Population on a Remote Island.

The reduced species richness typical of oceanic islands provides an interesting environmental setup to examine in natura the epidemiological dynamics of infectious agents with potential implications for public health and/or conservation. On Amsterdam Island (Indian Ocean), recurrent die-offs of Indian yellow-nosed albatross (Thalassarche carteri) nestlings have been attributed to avian cholera, caused by the bacterium Pasteurella multocida. In order to help implementing efficient measures for the control of this disease, it is critical to better understand the local epidemiology of P. multocida and to examine its inter- and intra-annual infection dynamics. We evaluated the infection status of 264 yellow-nosed albatrosses over four successive breeding seasons using a real-time PCR targeting P. multocida DNA from cloacal swabs. Infection prevalence patterns revealed an intense circulation of P. multocida throughout the survey, with a steady but variable increase in infection prevalence within each breeding season. These epizootics were associated with massive nestling dies-offs, inducing very low fledging successes (≤ 20%). These results suggest important variations in the transmission dynamics of this pathogen. These findings and the developed PCR protocol have direct applications to guide future research and refine conservation plans aiming at controlling the disease.

Red coloration varies with dietary carotenoid access and nutritional condition in kittiwakes.

Carotenoid-based ornaments are common signaling features in animals. Although the mechanisms that link color-based signals to individual condition is key to understanding the evolution and function of these ornaments, they are most often poorly known. Several hypotheses have been posited. They include: (i) the role of foraging abilities on carotenoid acquisition and thereby carotenoid-based ornaments, and (ii) the role of internal processes linked to individual quality on the allocation and conversion of carotenoids in integuments. Here, we tested the influence of dietary carotenoid access versus internal process on gape coloration in black-legged kittiwakes (Rissa tridactyla). This seabird displays a vibrant red gape, whose coloration varies with individual quality in males and is due to the deposition of red ketocarotenoids, such as astaxanthin. We decreased hydroxycarotenoid and ketocarotenoid levels in plasma, but increased efficiency in internal processes linked to nutritional condition, by supplementing breeding males with capelin, a natural energy-rich fish prey. We found that, despite having lower carotenoid levels in plasma, supplemented birds developed redder coloration than control birds, but only in the year when dietary levels of astaxanthin in the natural diet were low. In contrast, in the astaxanthin-rich year, supplemented males had a less-red gape than unsupplemented birds. These results suggest that inter-individual differences in internal processes may be sufficient to maintain the honesty of gape coloration under conditions of low dietary astaxanthin levels. Nonetheless, when inter-individual variations in dietary astaxanthin levels are elevated (such as in the crustacean-rich year), carotenoid access seems a more limiting factor to the expression of gape coloration than internal processes. Therefore, our study revealed a complex mechanism of gape color production in kittiwakes, and suggests that the main factor maintaining the condition dependency of this ornaments may vary with environmental conditions and diet composition.

Exposure of breeding albatrosses to the agent of avian cholera: dynamics of antibody levels and ecological implications.

Despite critical implications for disease dynamics and surveillance in wild long-lived species, the immune response after exposure to potentially highly pathogenic bacterial disease agents is still poorly known. Among infectious diseases threatening wild populations, avian cholera, caused by the bacterium Pasteurella multocida, is a major concern. It frequently causes massive mortality events in wild populations, notably affecting nestlings of Indian yellow-nosed albatrosses (Thalassarche carteri) in the Indian Ocean. If adults are able to mount a long-term immune response, this could have important consequences regarding the dynamics of the pathogen in the local host community and the potential interest of vaccinating breeding females to transfer immunity to their offspring. By tracking the dynamics of antibodies against P. multocida during 4 years and implementing a vaccination experiment in a population of yellow-nosed albatrosses, we show that a significant proportion of adults were naturally exposed despite high annual survival for both vaccinated and non-vaccinated individuals. Adult-specific antibody levels were thus maintained long enough to inform about recent exposure. However, only low levels of maternal antibodies could be detected in nestlings the year following a vaccination of their mothers. A modification of the vaccine formulation and the possibility to re-vaccinate females 2 years after the first vaccination revealed that vaccines have the potential to elicit a stronger and more persistent response. Such results highlight the value of long-term observational and experimental studies of host exposure to infectious agents in the wild, where ecological and evolutionary processes are likely critical for driving disease dynamics.

Avian cholera outbreaks threaten seabird species on Amsterdam Island.

Infectious diseases may be particularly critical for the conservation of endangered species. A striking example is the recurrent outbreaks that have been occurring in seabirds on Amsterdam Island for the past 30 years, threatening populations of three Endangered seabird species and of the endemic, Critically Endangered Amsterdam albatross Diomedea amsterdamensis. The bacteria Pasteurella multocida (avian cholera causative agent), and to a lesser extent Erysipelothrix rhusiopathiae (erysipelas causative agent), were both suspected to be responsible for these epidemics. Despite this critical situation, demographic trends were not available for these threatened populations, and the occurrence and characterization of potential causative agents of epizootics remain poorly known. The aims of the current study were to (i) provide an update of population trends for four threatened seabird species monitored on Amsterdam Island, (ii) assess the occurrence of P. multocida, and E. rhusiopathiae in live birds from five species, (iii) search for other infectious agents in these samples and, (iv) isolate and genotype the causative agent(s) of epizooties from dead birds. Our study shows that the demographic situation has worsened substantially in three seabird species during the past decade, with extremely low reproductive success and declining populations for Indian yellow-nosed albatrosses Thalassarche carteri, sooty albatrosses Phoebetria fusca, and northern rockhopper penguins Eudyptes moseleyi. Pasteurella multocida or E. rhusiopathiae were detected by PCR in live birds of all five investigated species, while results were negative for eight additional infectious agents. A single strain of P. multocida was repeatedly cultured from dead birds, while no E. rhusiopathiae could be isolated. These results highlight the significance of P. multocida in this particular eco-epidemiological system as the main agent responsible for epizootics. The study stresses the urgent need to implement mitigation measures to alter the course of avian cholera outbreaks threatening the persistence of seabird populations on Amsterdam Island.

Blue petrels recognize the odor of their egg.

Most studies on avian olfactory communication have focused on mate choice, and the importance of olfaction in subsequent nesting stages has been poorly explored. In particular, the role of olfactory cues in egg recognition has received little attention, despite eggs potentially being spread with parental odorous secretions known to elicit individual discrimination. Here, we used behavioral choice tests to determine whether female blue petrels (Halobaena caerulea) can discriminate the odor of their own egg from the odor of a conspecific egg. Females preferentially approached the odor of their own egg, suggesting that blue petrels can recognize their own egg using odor cues. This finding raises the question of the adaptive value of this mechanism, and may inspire further research on odor-based egg discrimination in species suffering brood parasitism.

Adaptation of avian influenza virus to a swine host.

The emergence of pathogenic RNA viruses into new hosts can have dramatic consequences for both livestock and public health. Here we characterize the viral genetic changes that were observed in a previous study which experimentally adapted a field isolate of duck influenza virus to swine respiratory cells. Both pre-existing and de novo mutations were selected during this adaptation. We compare the in vitro growth dynamics of the adapted virus with those of the original strain as well as all possible reassortants using reverse genetics. This full factorial design showed that viral gene segments are involved in complex epistatic interactions on virus fitness, including negative and sign epistasis. We also identify two point mutations at positions 67 and 113 of the HA2 subunit of the hemagglutinin protein conferring a fast growth phenotype on the naïve avian virus in swine cells. These HA2 mutations enhance the pH dependent, HA-mediated membrane fusion. A global H1 maximum-likelihood phylogenetic analysis, combined with comprehensive ancestry reconstruction and tests for directional selection, confirmed the field relevance of the mutation at position 113 of HA2. Most notably, this mutation was associated with the establishment of the H1 'avian-like' swine influenza lineage, regarded as the most likely to cause the next influenza pandemic in humans. This multidisciplinary approach to study the genetics of viral adaptation provides unique insights on the underlying processes leading to influenza emergence in a new host species, and identifies specific targets for future surveillance and functional studies.

Range-wide parallel climate-associated genomic clines in Atlantic salmon.

Clinal variation across replicated environmental gradients can reveal evidence of local adaptation, providing insight into the demographic and evolutionary processes that shape intraspecific diversity. Using 1773 genome-wide single nucleotide polymorphisms we evaluated latitudinal variation in allele frequency for 134 populations of North American and European Atlantic salmon (Salmo salar). We detected 84 (4.74%) and 195 (11%) loci showing clinal patterns in North America and Europe, respectively, with 12 clinal loci in common between continents. Clinal single nucleotide polymorphisms were evenly distributed across the salmon genome and logistic regression revealed significant associations with latitude and seasonal temperatures, particularly average spring temperature in both continents. Loci displaying parallel clines were associated with several metabolic and immune functions, suggesting a potential basis for climate-associated adaptive differentiation. These climate-based clines collectively suggest evidence of large-scale environmental associated differences on either side of the North Atlantic. Our results support patterns of parallel evolution on both sides of the North Atlantic, with evidence of both similar and divergent underlying genetic architecture. The identification of climate-associated genomic clines illuminates the role of selection and demographic processes on intraspecific diversity in this species and provides a context in which to evaluate the impacts of climate change.

Migration, Prospecting, Dispersal? What Host Movement Matters for Infectious Agent Circulation?

Spatial disease ecology is emerging as a new field that requires the integration of complementary approaches to address how the distribution and movements of hosts and parasites may condition the dynamics of their interactions. In this context, migration, the seasonal movement of animals to different zones of their distribution, is assumed to play a key role in the broad scale circulation of parasites and pathogens. Nevertheless, migration is not the only type of host movement that can influence the spatial ecology, evolution, and epidemiology of infectious diseases. Dispersal, the movement of individuals between the location where they were born or bred to a location where they breed, has attracted attention as another important type of movement for the spatial dynamics of infectious diseases. Host dispersal has notably been identified as a key factor for the evolution of host-parasite interactions as it implies gene flow among local host populations and thus can alter patterns of coevolution with infectious agents across spatial scales. However, not all movements between host populations lead to dispersal per se. One type of host movement that has been neglected, but that may also play a role in parasite spread is prospecting, i.e., movements targeted at selecting and securing new habitat for future breeding. Prospecting movements, which have been studied in detail in certain social species, could result in the dispersal of infectious agents among different host populations without necessarily involving host dispersal. In this article, we outline how these various types of host movements might influence the circulation of infectious disease agents and discuss methodological approaches that could be used to assess their importance. We specifically focus on examples from work on colonial seabirds, ticks, and tick-borne infectious agents. These are convenient biological models because they are strongly spatially structured and involve relatively simple communities of interacting species. Overall, this review emphasizes that explicit consideration of the behavioral and population ecology of hosts and parasites is required to disentangle the relative roles of different types of movement for the spread of infectious diseases.

Transatlantic secondary contact in Atlantic Salmon, comparing microsatellites, a single nucleotide polymorphism array and restriction-site associated DNA sequencing for the resolution of complex spatial structure.

Identification of discrete and unique assemblages of individuals or populations is central to the management of exploited species. Advances in population genomics provide new opportunities for re-evaluating existing conservation units but comparisons among approaches remain rare. We compare the utility of RAD-seq, a single nucleotide polymorphism (SNP) array and a microsatellite panel to resolve spatial structuring under a scenario of possible trans-Atlantic secondary contact in a threatened Atlantic Salmon, Salmo salar, population in southern Newfoundland. Bayesian clustering indentified two large groups subdividing the existing conservation unit and multivariate analyses indicated significant similarity in spatial structuring among the three data sets. mtDNA alleles diagnostic for European ancestry displayed increased frequency in southeastern Newfoundland and were correlated with spatial structure in all marker types. Evidence consistent with introgression among these two groups was present in both SNP data sets but not the microsatellite data. Asymmetry in the degree of introgression was also apparent in SNP data sets with evidence of gene flow towards the east or European type. This work highlights the utility of RAD-seq based approaches for the resolution of complex spatial patterns, resolves a region of trans-Atlantic secondary contact in Atlantic Salmon in Newfoundland and demonstrates the utility of multiple marker comparisons in identifying dynamics of introgression.

Intra-host viral variability in children clinically infected with H1N1 (2009) pandemic influenza.

Recent in-depth genetic analyses of influenza A virus samples have revealed patterns of intra-host viral genetic variability in a variety of relevant systems. These have included laboratory infected poultry, horses, pigs, chicken eggs and swine respiratory cells, as well as naturally infected poultry and horses. In humans, next generation sequencing techniques have enabled the study of genetic variability at specific positions of the viral genome. The present study investigated how 454 pyrosequencing could help unravel intra-host genetic diversity patterns on the full-length viral hæmagglutinin and neuraminidase genes from human H1N1 (2009) pandemic influenza clinical cases. This approach revealed unexpected patterns of co-infection in a 3-week old toddler, arising from rapid and complex reassortment phenomena on a local epidemiological scale. It also suggested the possible existence of very low frequency mutants resistant to neuraminidase inhibitors in two untreated patients. As well as revealing patterns of intra-host viral variability, this report highlights technical challenges in the appraisal of scientifically and medically relevant topics such as the natural occurrence of homologous recombination or very low frequency drug-resistant variants in influenza virus populations.

Conservation genomics of anadromous Atlantic salmon across its North American range: outlier loci identify the same patterns of population structure as neutral loci.

Anadromous Atlantic salmon (Salmo salar) is a species of major conservation and management concern in North America, where population abundance has been declining over the past 30 years. Effective conservation actions require the delineation of conservation units to appropriately reflect the spatial scale of intraspecific variation and local adaptation. Towards this goal, we used the most comprehensive genetic and genomic database for Atlantic salmon to date, covering the entire North American range of the species. The database included microsatellite data from 9142 individuals from 149 sampling locations and data from a medium-density SNP array providing genotypes for >3000 SNPs for 50 sampling locations. We used neutral and putatively selected loci to integrate adaptive information in the definition of conservation units. Bayesian clustering with the microsatellite data set and with neutral SNPs identified regional groupings largely consistent with previously published regional assessments. The use of outlier SNPs did not result in major differences in the regional groupings, suggesting that neutral markers can reflect the geographic scale of local adaptation despite not being under selection. We also performed assignment tests to compare power obtained from microsatellites, neutral SNPs and outlier SNPs. Using SNP data substantially improved power compared to microsatellites, and an assignment success of 97% to the population of origin and of 100% to the region of origin was achieved when all SNP loci were used. Using outlier SNPs only resulted in minor improvements to assignment success to the population of origin but improved regional assignment. We discuss the implications of these new genetic resources for the conservation and management of Atlantic salmon in North America.