Genomic advances in the study of the mosquito vector during avian malaria infection.

Invertebrate host­parasite associations are one of the keystones in order to understand vector-borne diseases. The study of these specific interactions provides information not only about how the vector is affected by the parasite at the gene-expression level, but might also reveal mosquito strategies for blocking the transmission of the parasites. A very well-known vector for human malaria is Anopheles gambiae. This mosquito species has been the main focus for genomics studies determining essential key genes and pathways over the course of a malaria infection. However, to-date there is an important knowledge gap concerning other non-mammophilic mosquito species, for example some species from the Culex genera which may transmit avian malaria but also zoonotic pathogens such as West Nile virus. From an evolutionary perspective, these 2 mosquito genera diverged 170 million years ago, hence allowing studies in both species determining evolutionary conserved genes essential during malaria infections, which in turn might help to find key genes for blocking malaria cycle inside the mosquito. Here, we extensively review the current knowledge on key genes and pathways expressed in Anopheles over the course of malaria infections and highlight the importance of conducting genomic investigations for detecting pathways in Culex mosquitoes linked to infection of avian malaria. By pooling this information, we underline the need to increase genomic studies in mosquito­parasite associations, such as the one in Culex­Plasmodium, that can provide a better understanding of the infection dynamics in wildlife and reduce the negative impact on ecosystems.

Seasonal variations of intensity of avian malaria infection in the Thousand Island Lake System, China.

BACKGROUND: Migratory birds play an important part in the spread of parasites, with more or less impact on resident birds. Previous studies focus on the prevalence of parasites, but changes in infection intensity over time have rarely been studied. As infection intensity can be quantified by qPCR, we measured infection intensity during different seasons, which is important for our understanding of parasite transmission mechanisms. METHODS: Wild birds were captured at the Thousand Island Lake with mist nets and tested for avian hemosporidiosis infections using nested PCR. Parasites were identified using the MalAvi database. Then, we used qPCR to quantify the infection intensity. We analyzed the monthly trends of intensity for all species and for different migratory status, parasite genera and sexes. RESULTS: Of 1101 individuals, 407 were infected (37.0%) of which 95 were newly identified and mainly from the genus Leucocytozoon. The total intensity trend shows peaks at the start of summer, during the breeding season of hosts and during the over-winter season. Different parasite genera show different monthly trends. Plasmodium causes high prevalence and infection intensity of winter visitors. Female hosts show significant seasonal trends of infection intensity. CONCLUSIONS: The seasonal changes of infection intensity is consistent with the prevalence. Peaks occur early and during the breeding season and then there is a downward trend. Spring relapses and avian immunity are possible reasons that could explain this phenomenon. In our study, winter visitors have a higher prevalence and infection intensity, but they rarely share parasites with resident birds. This shows that they were infected with Plasmodium during their departure or migration and rarely transmit the disease to resident birds. The different infection patterns of different parasite species may be due to vectors or other ecological properties.

Immune gene expression in the mosquito vector Culex quinquefasciatus during an avian malaria infection.

Plasmodium relictum is the most widespread avian malaria parasite in the world. It is listed as one of the 100 most dangerous invasive species, having been responsible for the extinction of several endemic bird species, and the near-demise of several others. Here we present the first transcriptomic study focused on the effect of P. relictum on the immune system of its vector (the mosquito Culex quinquefasciatus) at different times post-infection. We show that over 50% of immune genes identified as being part of the Toll pathway and 30%-40% of the immune genes identified within the Imd pathway are overexpressed during the critical period spanning the parasite's oocyst and sporozoite formation (8-12 days), revealing the crucial role played by both these pathways in this natural mosquito-Plasmodium combination. Comparison of infected mosquitoes with their uninfected counterparts also revealed some unexpected immune RNA expression patterns earlier and later in the infection: significant differences in expression of several immune effectors were observed as early as 30 min after ingestion of the infected blood meal. In addition, in the later stages of the infection (towards the end of the mosquito lifespan), we observed an unexpected increase in immune investment in uninfected, but not in infected, mosquitoes. In conclusion, our work extends the comparative transcriptomic analyses of malaria-infected mosquitoes beyond human and rodent parasites and provides insights into the degree of conservation of immune pathways and into the selective pressures exerted by Plasmodium parasites on their vectors.

Genomic sequence capture of Plasmodium relictum in experimentally infected birds.

BACKGROUND: Sequencing parasite genomes in the presence of host DNA is challenging. Sequence capture can overcome this problem by using RNA probes that hybridize with the parasite DNA and then are removed from solution, thus isolating the parasite DNA for efficient sequencing. METHODS: Here we describe a set of sequence capture probes designed to target 1035 genes (c. 2.5 Mbp) of the globally distributed avian haemosporidian parasite, Plasmodium relictum. Previous sequence capture studies of avian haemosporidians from the genus Haemoproteus have shown that sequencing success depends on parasitemia, with low-intensity, chronic infections (typical of most infected birds in the wild) often being difficult to sequence. We evaluate the relationship between parasitemia and sequencing success using birds experimentally infected with P. relictum and kept under laboratory conditions. RESULTS: We confirm the dependence of sequencing success on parasitemia. Sequencing success was low for birds with low levels of parasitemia (< 1% infected red blood cells) and high for birds with higher levels of parasitemia. Plasmodium relictum is composed of multiple lineages defined by their mitochondrial DNA haplotype including three that are widespread (SGS1, GRW11, and GRW4); the probes successfully isolated DNA from all three. Furthermore, we used data from 25 genes to describe both among- and within-lineage genetic variation. For example, two samples of SGS1 isolated from different host species differed by 11 substitutions across those 25 genes. CONCLUSIONS: The sequence capture approach we describe will allow for the generation of genomic data that will contribute to our understanding of the population genetic structure and evolutionary history of P. relictum, an extreme host generalist and widespread parasite.

A comparative analysis of the dynamics of Plasmodium relictum (GRW4) development in the blood during single and co-infections.

Although co-infections and interactions of parasites are a very common phenomenon in the wild, information received from studies on avian Plasmodium spp. is scarce and fragmented due to its complex nature. Different interactions of parasites and domination of one parasite may have a detrimental effect on transmission success of another pathogen. Untangling these interactions and competitive behavior of malarial parasites may help understanding why some haemosporidian parasites are dominant in certain host species, while others are observed only occasionally. We investigated the development of Plasmodium relictum (genetic lineage GRW4) during single and co-infection with a closely related lineage SGS1, with the aim to determine whether co-infections affect parasite development and condition of experimentally infected Eurasian siskins (Spinus spinus). For the experimental study of these two closely related lineages, a new qPCR protocol was designed to accurately quantify the parasitemia, i.e. the amount of infected red blood cells, during the blood stages of each of the lineages. Our results show that during co-infection, GRW4 parasitemia was transient and disappeared from peripheral blood during acute increases of SGS1. Health parameters of infected birds did not differ between the GRW4 single infected group and the co-infection group. GRW4 induced infection was outcompeted and suppressed by the presence of the lineage SGS1, which is broadly transmitted in Northern Europe. This suggests that double infections and dominating lineages in the area may influence the transmission success of some avian Plasmodium parasites.

Low MSP-1 haplotype diversity in the West Palearctic population of the avian malaria parasite Plasmodium relictum.

BACKGROUND: Although avian Plasmodium species are widespread and common across the globe, limited data exist on how genetically variable their populations are. Here, the hypothesis that the avian blood parasite Plasmodium relictum exhibits very low genetic diversity in its Western Palearctic transmission area (from Morocco to Sweden in the north and Transcaucasia in the east) was tested. METHODS: The genetic diversity of Plasmodium relictum was investigated by sequencing a portion (block 14) of the fast-evolving merozoite surface protein 1 (MSP1) gene in 75 different P. relictum infections from 36 host species. Furthermore, the full-length MSP1 sequences representing the common block 14 allele was sequenced in order to investigate if additional variation could be found outside block 14. RESULTS: The majority (72 of 75) of the sequenced infections shared the same MSP1 allele. This common allele has previously been found to be the dominant allele transmitted in Europe. CONCLUSION: The results corroborate earlier findings derived from a limited dataset that the globally transmitted malaria parasite P. relictum exhibits very low genetic diversity in its Western Palearctic transmission area. This is likely the result of a recent introduction event or a selective sweep.

Gene regulation of the avian malaria parasite Plasmodium relictum, during the different stages within the mosquito vector.

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

Shifts in gene expression variability in the blood-stage of Plasmodium relictum.

Avian malaria is a common and widespread disease of birds caused by a diverse group of pathogens of the genera Plasmodium. We investigated the transcriptomal profiles of one of the most common species, Plasmodium relictum, lineage SGS1, at multiple timepoints during the blood stages of the infection under experimental settings. The parasite showed well separated overall transcriptome profiles between day 8 and 20 after the infection, shown by well separated PCA profiles. Moreover, gene expression becomes more heterogenous within the experimental group late in the infection, either due to adaptations to individual differences between the experimental hosts, or due to desynchronisation of the life-cycle of the parasite. Overall, this study shows how the avian malaria system can be used to study gene expression of the avian Plasmodium parasite under controlled experimental settings, thus allowing for future comparative analysis of gene responses of parasite with different life-history traits and host effects.

Early-life gut dysbiosis linked to juvenile mortality in ostriches.

BACKGROUND: Imbalances in the gut microbial community (dysbiosis) of vertebrates have been associated with several gastrointestinal and autoimmune diseases. However, it is unclear which taxa are associated with gut dysbiosis, and if particular gut regions or specific time periods during ontogeny are more susceptible. We also know very little of this process in non-model organisms, despite an increasing realization of the general importance of gut microbiota for health. METHODS: Here, we examine the changes that occur in the microbiome during dysbiosis in different parts of the gastrointestinal tract in a long-lived bird with high juvenile mortality, the ostrich (Struthio camelus). We evaluated the 16S rRNA gene composition of the ileum, cecum, and colon of 68 individuals that died of suspected enterocolitis during the first 3 months of life (diseased individuals), and of 50 healthy individuals that were euthanized as age-matched controls. We combined these data with longitudinal environmental and fecal sampling to identify potential sources of pathogenic bacteria and to unravel at which stage of development dysbiosis-associated bacteria emerge. RESULTS: Diseased individuals had drastically lower microbial alpha diversity and differed substantially in their microbial beta diversity from control individuals in all three regions of the gastrointestinal tract. The clear relationship between low diversity and disease was consistent across all ages in the ileum, but decreased with age in the cecum and colon. Several taxa were associated with mortality (Enterobacteriaceae, Peptostreptococcaceae, Porphyromonadaceae, Clostridium), while others were associated with health (Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, Turicibacter, Roseburia). Environmental samples showed no evidence of dysbiosis-associated bacteria being present in either the food, water, or soil substrate. Instead, the repeated fecal sampling showed that pathobionts were already present shortly after hatching and proliferated in individuals with low microbial diversity, resulting in high mortality several weeks later. CONCLUSIONS: Identifying the origins of pathobionts in neonates and the factors that subsequently influence the establishment of diverse gut microbiota may be key to understanding dysbiosis and host development. Video Abstract.

Host Transcriptional Responses to High- and Low-Virulent Avian Malaria Parasites.

The transcriptional response of hosts to genetically similar pathogens can vary substantially, with important implications for disease severity and host fitness. A low pathogen load can theoretically elicit both high and low host responses, as the outcome depends on both the effectiveness of the host at suppressing the pathogen and the ability of the pathogen to evade the immune system. Here, we investigate the transcriptional response of Eurasian siskins (Spinus spinus) to two closely related lineages of the malaria parasite Plasmodium relictum. Birds were infected with either the high-virulent lineage P. relictum SGS1, the low-virulent sister lineage P. relictum GRW4, or sham-injected (controls). Blood samples for RNA sequencing were collected at four time points during the course of infection, totaling 76 transcriptomes from 19 birds. Hosts infected with SGS1 experienced up to 87% parasitemia and major transcriptome shifts throughout the infection, and multiple genes showed strong correlation with parasitemia. In contrast, GRW4-infected hosts displayed low parasitemia (maximum 0.7%) with a minor transcriptional response. We furthermore demonstrate that the baseline gene expression levels of hosts prior to infection were irrelevant as immunocompetence markers, as they could not predict future pathogen load. This study shows that the magnitude of the host transcriptional response can differ markedly from related parasites with different virulence, and it enables a better understanding of the molecular interactions taking place between hosts and parasites.

Within-Lineage Divergence of Avian Haemosporidians: A Case Study to Reveal the Origin of a Widespread Haemoproteus Parasite.

Avian haemosporidian parasites are particularly diverse and widespread. To date, more than 3,000 distinct cytochrome b lineages have been recorded, of which some present extremely wide geographical distributions, even including multiple continents. Whether these isolates represent one or several cryptic species remains unknown. Here we carried out a case study of SISKIN1, a common haemosporidian parasite lineage belonging to the morphologically described species Haemoproteus tartakovskyi. To shed light on its evolutionary origin, we investigated the divergence between SISKIN1 isolates obtained from siskins and redpolls in Europe (Russia and Sweden) and house finches in North America (Mexico). First, we used sequence capture of a small data set (2 Russian isolates and 1 Mexican isolate) to investigate the genetic structure based on the full-length mitochondrial genome and ∼1,000 genes. The mitochondrial genomes of Russian isolates were identical with each other but differed from the Mexican one at 6 positions. The nuclear divergence between Russian and Mexican isolates was on average 2.8%, close to what has been observed between 2 species of malaria parasites that respectively infect humans (Plasmodium falciparum) and gorillas (Plasmodium praefalciparum). Second, we used the expanded data set (15 samples in total) to investigate the genetic structure in 3 genes known to be involved in host invasion. The European isolates were identical across all sequenced genes, whereas the Mexican isolates were highly diverse. The lack of shared alleles between European and Mexican populations suggests that they might have diverged in isolation without gene flow. From the MalAvi database we examined the lineages most similar to the SISKIN1 barcode fragment (part of the cyt b gene) and found that most of them had been recorded in North and South America. This suggests that the lineage SISKIN1 originated in North America and subsequently spread to Europe. Our analyses support that the cyt b gene barcoding region is a useful marker for identification of avian haemosporidian lineages that can classify them into clusters of closely related parasites, but to further investigate species limits and evolutionary history, molecular data from multiple faster-evolving genes are required.

Major shifts in gut microbiota during development and its relationship to growth in ostriches.

The development of gut microbiota during ontogeny is emerging as an important process influencing physiology, immunity and fitness in vertebrates. However, knowledge of how bacteria colonize the juvenile gut, how this is influenced by changes in the diversity of gut bacteria and to what extent this influences host fitness, particularly in nonmodel organisms, is lacking. Here we used 16S rRNA gene sequencing to describe the successional development of the faecal microbiome in ostriches (Struthio camelus, n = 66, repeatedly sampled) over the first 3 months of life and its relationship to growth. We found a gradual increase in microbial diversity with age that involved multiple colonization and extinction events and a major taxonomic shift in bacteria that coincided with the cessation of yolk absorption. Comparisons with the microbiota of adults (n = 5) revealed that the chicks became more similar in their microbial diversity and composition to adults as they aged. There was a five-fold difference in juvenile growth during development, and growth during the first week of age was strongly positively correlated with the abundance of the genus Bacteroides and negatively correlated with Akkermansia. After the first week, the abundances of six phylogenetically diverse families (Peptococcaceae, S24-7, Verrucomicrobiae, Anaeroplasmataceae, Streptococcaceae, Methanobacteriaceae) were associated with subsequent reductions in chick growth in an age-specific and transient manner. These results have broad implications for our understanding of the development of gut microbiota and its associations with animal growth.

The success of sequence capture in relation to phylogenetic distance from a reference genome: a case study of avian haemosporidian parasites.

Genomic sequencing of avian haemosporidian parasites (Haemosporida) has been challenging due to excessive contamination from host DNA. In this study, we developed a cost-effective protocol to obtain parasite sequences from naturally infected birds, based on targeted sequence capture and next generation sequencing. With the genomic data of Haemoproteus tartakovskyi as a reference, we successfully sequenced up to 1000 genes from each of the 15 selected samples belonging to nine different cytochrome b lineages, eight of which belong to Haemoproteus and one to Plasmodium. The targeted sequences were enriched to ∼104-fold, and mixed infections were identified as well as the proportions of each mixed lineage. We found that the total number of reads and the proportions of exons sequenced decreased when the parasite lineage became more divergent from the reference genome. For each of the samples, the recovery of sequences from different exons varied with the function and GC content of the exon. From the obtained sequences, we detected within-lineage variation in both mitochondrial and nuclear genes, which may be a result of local adaptation to different host species and environmental conditions. This targeted sequence capture protocol can be applied to a broader range of species and will open a new door for further studies on disease diagnostics and comparative analysis of haemosporidians evolution.

De novo synthesis of thiamine (vitamin B1) is the ancestral state in Plasmodium parasites - evidence from avian haemosporidians.

Parasites often have reduced genomes as their own genes become redundant when utilizing their host as a source of metabolites, thus losing their own de novo production of metabolites. Primate malaria parasites can synthesize vitamin B1 (thiamine) de novo but rodent malaria and other genome-sequenced apicomplexans cannot, as the three essential genes responsible for this pathway are absent in their genomes. The unique presence of functional thiamine synthesis genes in primate malaria parasites and their sequence similarities to bacterial orthologues, have led to speculations that this pathway was horizontally acquired from bacteria. Here we show that the genes essential for the de novo synthesis of thiamine are found also in avian Plasmodium species. Importantly, they are also present in species phylogenetically basal to all mammalian and avian Plasmodium parasites, i.e. Haemoproteus. Furthermore, we found that these genes are expressed during the blood stage of the avian malaria infection, indicating that this metabolic pathway is actively transcribed. We conclude that the ability to synthesize thiamine is widespread among haemosporidians, with a recent loss in the rodent malaria species.

The transcriptome of the avian malaria parasite Plasmodium ashfordi displays host-specific gene expression.

Malaria parasites (Plasmodium spp.) include some of the world's most widespread and virulent pathogens. Our knowledge of the molecular mechanisms these parasites use to invade and exploit their hosts other than in mice and primates is, however, extremely limited. It is therefore imperative to characterize transcriptome-wide gene expression from nonmodel malaria parasites and how this varies across individual hosts. Here, we used high-throughput Illumina RNA sequencing on blood from wild-caught Eurasian siskins experimentally infected with a clonal strain of the avian malaria parasite Plasmodium ashfordi (lineage GRW2). Using a bioinformatic multistep approach to filter out host transcripts, we successfully assembled the blood-stage transcriptome of P. ashfordi. A total of 11 954 expressed transcripts were identified, and 7860 were annotated with protein information. We quantified gene expression levels of all parasite transcripts across three hosts during two infection stages - peak and decreasing parasitemia. Interestingly, parasites from the same host displayed remarkably similar expression profiles during different infection stages, but showed large differences across hosts, indicating that P. ashfordi may adjust its gene expression to specific host individuals. We further show that the majority of transcripts are most similar to the human parasite Plasmodium falciparum, and a large number of red blood cell invasion genes were discovered, suggesting evolutionary conserved invasion strategies between mammalian and avian Plasmodium. The transcriptome of P. ashfordi and its host-specific gene expression advances our understanding of Plasmodium plasticity and is a valuable resource as it allows for further studies analysing gene evolution and comparisons of parasite gene expression.

The Evolution of Innate Immune Genes: Purifying and Balancing Selection on ß-Defensins in Waterfowl.

In disease dynamics, high immune gene diversity can confer a selective advantage to hosts in the face of a rapidly evolving and diverse pathogen fauna. This is supported empirically for genes involved in pathogen recognition and signalling. In contrast, effector genes involved in pathogen clearance may be more constrained. ß-Defensins are innate immune effector genes; their main mode of action is via disruption of microbial membranes. Here, five ß-defensin genes were characterized in mallards (Anas platyrhynchos) and other waterfowl; key reservoir species for many zoonotic diseases. All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific ß-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated ß-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of ß-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian ß-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.

The Genome of Haemoproteus tartakovskyi and Its Relationship to Human Malaria Parasites.

The phylogenetic relationships among hemosporidian parasites, including the origin of Plasmodium falciparum, the most virulent malaria parasite of humans, have been heavily debated for decades. Studies based on multiple-gene sequences have helped settle many of these controversial phylogenetic issues. However, denser taxon sampling and genome-wide analyses are needed to confidently resolve the evolutionay relationships among hemosporidian parasites. Genome sequences of several Plasmodium parasites are available but only for species infecting primates and rodents. To root the phylogenetic tree of Plasmodium, genomic data from related parasites of birds or reptiles are required. Here, we use a novel approach to isolate parasite DNA from microgametes and describe the first genome of a bird parasite in the sister genus to Plasmodium, Haemoproteus tartakovskyi Similar to Plasmodium parasites, H. tartakovskyi has a small genome (23.2 Mb, 5,990 genes) and a GC content (25.4%) closer to P. falciparum (19.3%) than to Plasmodium vivax (42.3%). Combined with novel transcriptome sequences of the bird parasite Plasmodium ashfordi, our phylogenomic analyses of 1,302 orthologous genes demonstrate that mammalian-infecting malaria parasites are monophyletic, thus rejecting the repeatedly proposed hypothesis that the ancestor of Laverania parasites originated from a secondary host shift from birds to humans. Genes and genomic features previously found to be shared between P. falciparum and bird malaria parasites, but absent in other mammal malaria parasites, are therefore signatures of maintained ancestral states. We foresee that the genome of H. tartakovskyi will open new directions for comparative evolutionary analyses of malarial adaptive traits.

Population genetic structure, differentiation, and diversity in Tetrix subulata pygmy grasshoppers: roles of population size and immigration.

Genetic diversity within and among populations and species is influenced by complex demographic and evolutionary processes. Despite extensive research, there is no consensus regarding how landscape structure, spatial distribution, gene flow, and population dynamics impact genetic composition of natural populations. Here, we used amplified fragment length polymorphisms (AFLPs) to investigate effects of population size, geographic isolation, immigration, and gene flow on genetic structure, divergence, and diversity in populations of Tetrix subulata pygmy grasshoppers (Orthoptera: Tetrigidae) from 20 sampling locations in southern Sweden. Analyses of 1564 AFLP markers revealed low to moderate levels of genetic diversity (PPL = 59.5-90.1; Hj = 0.23-0.32) within and significant divergence among sampling localities. This suggests that evolution of functional traits in response to divergent selection is possible and that gene flow is restricted. Genetic diversity increased with population size and with increasing proportion of long-winged phenotypes (a proxy of recent immigration) across populations on the island of Öland, but not on the mainland. Our data further suggested that the open water separating Öland from the mainland acts as a dispersal barrier that restricts migration and leads to genetic divergence among regions. Isolation by distance was evident for short interpopulation distances on the mainland, but gradually disappeared as populations separated by longer distances were included. Results illustrate that integrating ecological and molecular data is key to identifying drivers of population genetic structure in natural populations. Our findings also underscore the importance of landscape structure and spatial sampling scheme for conclusions regarding the role of gene flow and isolation by distance.

Detecting transmission areas of malaria parasites in a migratory bird species.

The identification of the regions where vector-borne diseases are transmitted is essential to study transmission patterns and to recognize future changes in environmental conditions that may potentially influence the transmission areas. SGS1, one of the lineages of Plasmodium relictum, is known to have active transmission in tropical Africa and temperate regions of Europe. Nuclear sequence data from isolates infected with SGS1 (based on merozoite surface protein 1 (MSP1) allelic diversity) have provided new insights on the distribution and transmission areas of these allelic variants. For example, MSP1 alleles transmitted in Africa differ from those transmitted in Europe, suggesting the existence of two populations of SGS1 lineages. However, no study has analysed the distribution of African and European transmitted alleles in Afro-Palearctic migratory birds. With this aim, we used a highly variable molecular marker to investigate whether juvenile house martins become infected in Europe before their first migration to Africa. We explored the MSP1 allelic diversity of P. relictum in adult and juvenile house martins. We found that juveniles were infected with SGS1 during their first weeks of life, confirming active transmission of SGS1 to house martins in Europe. Moreover, we found that all the juveniles and most of adults were infected with one European transmitted MSP1 allele, whereas two adult birds were infected with two African transmitted MSP1 alleles. These findings suggest that house martins are exposed to different strains of P. relictum in their winter and breeding quarters.

The avian transcriptome response to malaria infection.

Malaria parasites are highly virulent pathogens which infect a wide range of vertebrates. Despite their importance, the way different hosts control and suppress malaria infections remains poorly understood. With recent developments in next-generation sequencing techniques, however, it is now possible to quantify the response of the entire transcriptome to infections. We experimentally infected Eurasian siskins (Carduelis spinus) with avian malaria parasites (Plasmodium ashfordi), and used high-throughput RNA-sequencing to measure the avian transcriptome in blood collected before infection (day 0), during peak parasitemia (day 21 postinfection), and when parasitemia was decreasing (day 31). We found considerable differences in the transcriptomes of infected and uninfected individuals, with a large number of genes differentially expressed during both peak and decreasing parasitemia stages. These genes were overrepresented among functions involved in the immune system, stress response, cell death regulation, metabolism, and telomerase activity. Comparative analyses of the differentially expressed genes in our study to those found in other hosts of malaria (human and mouse) revealed a set of genes that are potentially involved in highly conserved evolutionary responses to malaria infection. By using RNA-sequencing we gained a more complete view of the host response, and were able to pinpoint not only well-documented host genes but also unannotated genes with clear significance during infection, such as microRNAs. This study shows how the avian blood transcriptome shifts in response to malaria infection, and we believe that it will facilitate further research into the diversity of molecular mechanisms that hosts utilize to fight malaria infections.