We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed the same trends of prevalence of Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%) and Leucocytozoon spp. (30% and 25%) in the same sample, testifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is relatively inexpensive and provides valuable information about directions how molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of relatively long duration of microscopy of each sample, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time-consuming.

We investigated the degree of geographical shifts of transmission areas of vector-borne avian blood parasites (Plasmodium, Haemoproteus and Leucocytozoon) over ecological and evolutionary timescales. Of 259 different parasite lineages obtained from 5886 screened birds sampled in Europe and Africa, only two lineages were confirmed to have current transmission in resident bird species in both geographical areas. We used a phylogenetic approach to show that parasites belonging to the genera Haemoproteus and Leucocytozoon rarely change transmission area and that these parasites are restricted to one resident bird fauna over a long evolutionary time span and are not freely spread between the continents with the help of migratory birds. Lineages of the genus Plasmodium seem more freely spread between the continents. We suggest that such a reduced transmission barrier of Plasmodium parasites is caused by their higher tendency to infect migratory bird species, which might facilitate shifting of transmission area. Although vector-borne parasites of these genera apparently can shift between a tropical and a temperate transmission area and these areas are linked with an immense amount of annual bird migration, our data suggest that novel introductions of these parasites into resident bird faunas are rather rare evolutionary events.

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.

Invasive species can displace natives, and thus identifying the traits that make aliens successful is crucial for predicting and preventing biodiversity loss. Pathogens may play an important role in the invasive process, facilitating colonization of their hosts in new continents and islands. According to the Novel Weapon Hypothesis, colonizers may out-compete local native species by bringing with them novel pathogens to which native species are not adapted. In contrast, the Enemy Release Hypothesis suggests that flourishing colonizers are successful because they have left their pathogens behind. To assess the role of avian malaria and related haemosporidian parasites in the global spread of a common invasive bird, we examined the prevalence and genetic diversity of haemosporidian parasites (order Haemosporida, genera Plasmodium and Haemoproteus) infecting house sparrows (Passer domesticus). We sampled house sparrows (N = 1820) from 58 locations on 6 continents. All the samples were tested using PCR-based methods; blood films from the PCR-positive birds were examined microscopically to identify parasite species. The results show that haemosporidian parasites in the house sparrows' native range are replaced by species from local host-generalist parasite fauna in the alien environments of North and South America. Furthermore, sparrows in colonized regions displayed a lower diversity and prevalence of parasite infections. Because the house sparrow lost its native parasites when colonizing the American continents, the release from these natural enemies may have facilitated its invasion in the last two centuries. Our findings therefore reject the Novel Weapon Hypothesis and are concordant with the Enemy Release Hypothesis.

Species of Haemoproteus (Haemosporida: Haemoproteidae), avian haemosporidians, have traditionally been described based on morphology of their gametocytes and on limited experimental information on their vertebrate host specificity. We investigated to what extent the morphological species are represented by monophyletic groups based on DNA sequence data using 2 different fragment lengths of the cytochrome b (cyt. b) gene. Phylogenetic reconstructions of obtained cyt. b lineages from 6 morphospecies of Haemoproteus showed that all lineages formed monophyletic clusters matching the morphospecies. Comparing our data with a recently published study showed that this is not always the case; the morphospecies H. belopolskyi consists of 2 distinct clusters of lineages that apparently have converged in morphology. However, the overall broad congruence between the molecular and morphological clustering of lineages will facilitate the integration of the knowledge obtained by traditional and molecular parasitology. Mean between morphospecies variation was 10-fold higher than the within species variation (5.5% vs. 0.54%), suggesting that Haemoproteus lineages with a genetic differentiation >5% are expected to be morphologically differentiated in most cases. When investigate the utility of 2 different fragment sizes of the cyt. b gene, the partial, 479-bp, cyt. b protocol picked up all mitochondrial (mt)DNA lineages that are found when using the full cyt. b gene, 1073 bp, suggesting that this protocol is sufficient for identification of most mtDNA lineages. All of the mtDNA lineages were associated with unique alleles when amplification was possible at a nuclear locus, strengthening the hypothesis that the designation of lineages based on mtDNA is largely genome-wide representative. We, therefore, propose the use of a cyt. b fragment of this length as a standard gene fragment for a DNA bar-coding system for avian Haemoproteus species.

BackgroundSympatric speciation—the divergence of populations into new species in absence of geographic barriers to hybridization—is the most debated mode of diversification of life forms. Parasitic organisms are prominent models for sympatric speciation, because they may colonise new hosts within the same geographic area and diverge through host specialization. However, it has been argued that this mode of parasite divergence is not strict sympatric speciation, because host shifts likely cause the sudden effective isolation of parasites, particularly if these are transmitted by vectors and therefore cannot select their hosts. Strict sympatric speciation would involve parasite lineages diverging within a single host species, without any population subdivision.Methodology/Principal FindingsHere we report a case of extraordinary divergence of sympatric, ecologically distinct, and reproductively isolated malaria parasites within a single avian host species, which apparently occurred without historical or extant subdivision of parasite or host populations.Conclusions/SignificanceThis discovery of within-host speciation changes our current view on the diversification potential of malaria parasites, because neither geographic isolation of host populations nor colonization of new host species are any longer necessary conditions to the formation of new parasite species.

A parasite's shift to a new host may have serious evolutionary consequences, since host switching usually is associated with a change in virulence and may lead to the evolution of emerging diseases. This phenomenon remains insufficiently studied in wildlife. Here, we combine microscopic examination of blood films and PCR-based methods to investigate the natural host specificity of Haemoproteus and Plasmodium spp. in birds of 4 families of the Passeriformes within a small geographic area. The material was collected on the Curonian Spit in the Baltic Sea between May and July in 2003-2004. A nested-PCR protocol was used for amplifying and sequencing a fragment of 480 nucleotides of the cytochrome b gene of the mtDNA of these parasites. Blood samples from 282 birds, which were positive both by microscopic examination of blood films and mtDNA amplification, were used in this study. We found that Haemoproteus majoris (lineages hPARUS1, hCCF5, hWW2, and hPHSIB1), Haemoproteus sp. (hWW1), Plasmodium (Haemamoeba) sp. (pSGS1), and Plasmodium (Haemamoeba) sp. (pGRW11) are capable of infecting birds belonging to different families of passeriform birds. Some species of Haemoproteus are less specific than have been traditionally believed. Haemoproteus majoris appears to have a genetic predisposition to have a broad host range. The level of host specificity varies markedly among different species of hemosporidian parasites of birds. The natural host range is thus not a reliable taxonomic character in the systematics of these parasites in the form in which it is still accepted in some recent taxonomic studies.

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.