Differential gene expression of Plasmodium homocircumflexum (lineage pCOLL4) across two experimentally infected passerine bird species.

Plasmodium parasites are present in a wide range of host species, some of which tend to be more susceptible than others, potentially as an outcome of evolved tolerance or resistance. Common starlings seem to cope with malaria infection while common crossbills are more susceptible to the same infections. That raises the question if the parasites rely on the same molecular mechanisms regardless of host species or do Plasmodium parasites change gene-expressions in accordance to the environment different hosts might provide? We used RNA-sequencing from starlings and crossbills, experimentally infected with Plasmodium homocircumflexum (lineage pCOLL4). The assembled transcriptome contained a total of 26,733 contigs. Parasite expression patterns differed between bird species. Parasites had higher expression of cell-invasion genes when infecting crossbills compared to starlings whereas in starlings genes related to apoptosis or/and oxidative stress showed higher expression levels. This article reveals how a Plasmodium parasite might adjust its expression and gene function depending on the host species infected.

Multiple cryptic species of sympatric generalists within the avian blood parasite Haemoproteus majoris.

The avian haemosporidian parasite Haemoproteus majoris has been reported to infect a wide range of passerine birds throughout the Holarctic ecozone. Five cytochrome b (cyt b) lineages have been described as belonging to the morphological species H. majoris, and these form a tight phylogenetic cluster together with 13 undescribed lineages that differ from each other by < 1.2% in sequence divergence. Records in a database (MalAvi) that contains global findings of haemosporidian lineages generated by universal primers suggest that these lineages vary substantially in host distribution. We confirm this pattern in a data set collected at Lake Kvismaren, Sweden, where three of the generalist lineages have local transmission. However, whether these lineages represent intraspecific mitochondrial diversity or clusters of cryptic species has previously not been examined. In this study, we developed novel molecular markers to amplify the partial segments of four nuclear genes to determine the level of genetic diversity and gene phylogenies among the five morphologically described cyt b lineages of H. majoris. All five cyt b lineages were strongly associated with unique nuclear alleles at all four nuclear loci, indicating that each mitochondrial lineage represents a distinct biological species. Within lineages, there was no apparent association between nuclear alleles and host species, indicating that they form genetically unstructured populations across multiple host species.

Circannual variation in blood parasitism in a sub-Saharan migrant passerine bird, the garden warbler.

Knowing the natural dynamics of pathogens in migratory birds is important, for example, to understand the factors that influence the transport of pathogens to and their transmission in new geographical areas, whereas the transmission of other pathogens might be restricted to a specific area. We studied haemosporidian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon in a migratory bird, the garden warbler Sylvia borin. Birds were sampled in spring, summer and early autumn at breeding grounds in Sweden, on migration at Capri, Italy and on arrival and departure from wintering staging areas in West Africa: mapping recoveries of garden warblers ringed in Fennoscandia and Capri showed that these sites are most probably on the migratory flyway of garden warblers breeding at Kvismaren. Overall, haemosporidian prevalence was 39%, involving 24 different parasite lineages. Prevalence varied significantly over the migratory cycle, with relatively high prevalence of blood parasites in the population on breeding grounds and at the onset of autumn migration, followed by marked declines in prevalence during migration both on spring and autumn passage. Importantly, we found that when examining circannual variation in the different lineages, significantly different prevalence profiles emerged both between and within genera. Our results suggest that differences in prevalence profiles are the result of either different parasite transmission strategies or coevolution between the host and the various parasite lineages. When separating parasites into common vs. rare lineages, we found that two peaks in the prevalence of rare parasites occur; on arrival at Swedish breeding grounds, and after the wintering period in Africa. Our results stress the importance of appropriate taxonomic resolution when examining host-parasite interactions, as variation in prevalence both between and within parasite genera can show markedly different patterns.

The genetic structure of Borrelia afzelii varies with geographic but not ecological sampling scale.

The genetic structure of a pathogen is an important determinant of its potential rate of adaptation and can thereby influence the dynamics of host-parasite interactions. We investigated how the genetic structure of Borrelia afzelii varies with geographic and ecological sampling scale. Genetic structure was measured as the degree of linkage disequilibrium (LD) across three loci. To test for the effects of geographic and ecological scale, we calculated LD across or within populations 4-82 km apart and across or within different mammal host species. There was highly significant LD across populations and host species. However, there was also evidence for genome-wide recombination, and the LD largely resulted from epidemic spread of certain haplotypes, rather than lack of recombination. Interestingly, the degree of LD was higher in each population than in the sample as a whole, i.e. LD increased with decreasing geographic scale. In contrast, there was no effect of ecological sampling scale on LD. Strong LD may impede the rate of adaptive evolution. Our results suggest this effect might be particularly strong at a small geographic scale.

In vitro tests of natural allelic variation of innate immune genes (avian ß-defensins) reveal functional differences in microbial inhibition.

Allelic variation in immune genes might result from, and contribute to, host-pathogen evolution. Functional allelic variation in the innate immune system has received little attention. Here, we investigate whether naturally occurring allelic variation within the avian innate immune system (ß-defensins) is associated with variation in antimicrobial activity. We tested differences in in vitro antimicrobial properties of the synthesized products of two alleles of avian ß-defensin 7, both of which occur at high frequency in natural populations of the great tit (Parus major). Only one allele strongly inhibited the growth of the gram-positive bacterium Staphylococcus aureus, but both alleles strongly inhibited growth of the gram-negative bacterium Escherechia coli. Our data demonstrate functional allelic variation in natural defensin genes, and we discuss how differences in efficacy against microbial species might contribute to maintaining this variation.

Parallelism and historical contingency during rapid ecotype divergence in an isopod.

Recent studies on parallel evolution have focused on the relative role of selection and historical contingency during adaptive divergence. Here, we study geographically separate and genetically independent lake populations of a freshwater isopod (Asellus aquaticus) in southern Sweden. In two of these lakes, a novel habitat was rapidly colonized by isopods from a source habitat. Rapid phenotypic changes in pigmentation, size and sexual behaviour have occurred, presumably in response to different predatory regimes. We partitioned the phenotypic variation arising from habitat ('selection': 81­94%), lake ('history': 0.1­6%) and lake × habitat interaction ('unique diversification': 0.4­13%) for several traits. There was a limited role for historical contingency but a strong signature of selection. We also found higher phenotypic variation in the source populations. Phenotype sorting during colonization and strong divergent selection might have contributed to these rapid changes. Consequently, phenotypic divergence was only weakly influenced by historical contingency.

Bird hosts, blood parasites and their vectors--associations uncovered by molecular analyses of blackfly blood meals.

The level of host specificity of blood-sucking invertebrates may have both ecological and evolutionary implications for the parasites they are transmitting. We used blood meals from wild-caught blackflies for molecular identification of parasites and hosts to examine patterns of host specificity and how these may affect the transmission of avian blood parasites of the genus Leucocytozoon. We found that five different species of ornithophilic blackflies preferred different species of birds when taking their blood meals. Of the blackflies that contained avian blood meals, 62% were infected with Leucocytozoon parasites, consisting of 15 different parasite lineages. For the blackfly species, there was a significant association between the host width (measured as the genetic differentiation between the used hosts) and the genetic similarity of the parasites in their blood meals. The absence of similar parasite in blood meals from blackflies with different host preferences is interpreted as a result of the vector-host associations. The observed associations between blackfly species and host species are therefore likely to hinder parasites to be transmitted between different host-groups, resulting in ecologically driven associations between certain parasite lineages and hosts species.

Locus-specific protocol for nine different innate immune genes (antimicrobial peptides: ß-defensins) across passerine bird species reveals within-species coding variation and a case of trans-species polymorphisms.

We present a locus-specific protocol suitable for the investigation, from extracted DNA, into natural inter- and intra-specific genetic variation in a group of nine innate immune genes, all belonging to the ß-defensin gene family. The products of these genes encode peptides with antimicrobial properties and form part of the innate immune system. The protocol amplifies the exon coding for the peptide that interacts with invading pathogens and is applicable across a wide range of passerine bird species, although with varying success depending on species. In several species tested, we found individuals to be heterozygous at several of the genes, highlighting the existence of coding genetic variation in this part of the immune system. Furthermore, for several of the genes, alleles have been conserved at the amino acid level across taxonomically distant bird species. In one case, we observed the existence of trans-species polymorphisms, often taken as evidence of balancing selection. The method will make it possible to investigate a part of the immune system for which there exists very little information of the genetic variation in wild vertebrate populations, thus making it possible to start investigating the selective forces under which the genes are evolving and the extent to which the found genetic variation is associated with pathogen susceptibility in wild populations.