Mihaela Ilieva

Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus

Little is known about the development of infection of malaria parasites of the genus Plasmodium in wild birds. We used qPCR, targeting specific mitochondrial lineages of Plasmodium ashfordi (GRW2) and Plasmodium relictum (GRW4), to monitor changes in intensities of parasitemia in captive great reed warblers Acrocephalus arundinaceus from summer to spring. The study involved both naturally infected adults and experimentally infected juveniles. The experiment demonstrated that P. ashfordi and P. relictum lineages differ substantially in several life-history traits (e.g. prepatent period and dynamics of parasitemia) and that individual hosts show substantial differences in responses to these infections. The intensity of parasitemia of lineages in mixed infections co-varied positively, suggesting a control mechanism by the host that is general across the parasite lineages. The intensity of parasitemia for individual hosts was highly repeatable suggesting variation between the host individuals in their genetic or acquired control of the infections. In future studies, care must be taken to avoid mixed infections in wild caught donors, and when possible use mosquitoes for the experiments as inoculation of infectious blood ignores important initial stages of the contact between the bird and the parasite.

Linkage between mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites, with a description of Plasmodium (Novyella) ashfordi sp. nov

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only seven morphospecies have been linked to these lineages. This study linked two molecular sequences with morphospecies of malaria parasites. Two species of Plasmodium (mitochondrial cytochrome b gene lineages P-GRW2 and P-GRW4) were isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to naive juvenile individuals of the same host species. Heavy parasitemia developed in the subinoculated birds, which enable identification of the species and deposition of their voucher specimens. Parasites of the lineage P-GRW2 were described as a new species, Plasmodium (Novyella) ashfordi, which is characterized primarily by the fan-like mature erythrocytic meronts containing seven to eight merozoites and the terminal position of clumped pigment granules in the gametocytes. Illustrations of the blood stages of the new species and Plasmodium (Haemamoeba) relictum (lineage P-GRW4) are given. The parasites of both lineages are transmitted in Africa and probably not in northern Europe. Other lineages closely related to P. ashfordi and P. relictum are identified. This study establishes the value of PCR-based identification of avian malaria parasites.

Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife

Haemosporidians (Haemosporida) are cosmopolitan in birds. Over 250 species of these blood parasites have been described and named; however, molecular markers remain unidentified for the great majority of them. This is unfortunate because linkage between DNA sequences and identifications based on morphological species can provide important information about patterns of transmission, virulence, and evolutionary biology of these organisms. There is an urgent need to remedy this because few experts possess the knowledge to identify haemosporidian species and few laboratories are involved in training these taxonomic skills. Here, we describe new mitochondrial cytochrome b markers for the polymerase chain reaction (PCR)-based detection of four widespread species of avian Haemoproteus (Haemoproteus hirundinis, Haemoproteus parabelopolskyi, Haemoproteus pastoris, Haemoproteus syrnii) and 1 species of Plasmodium (Plasmodium circumflexum). Illustrations of blood stages of the reported species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. This study indicates that morphological characters, which have been traditionally used in taxonomy of avian haemosporidian parasites, have a phylogenetic value. Perspectives on haemosporidian diagnostics using microscopic and PCR-based methods are discussed, particularly the difficulties in detection of light parasitemia, coinfections, and abortive parasite development. We emphasize that sensitive PCR amplifies more infections than can be transmitted; it should be used carefully in epidemiology studies, particularly in wildlife parasitology research. Because molecular studies are describing remarkably more parasite diversity than previously expected, the need for traditional taxonomy and traditional biological knowledge is becoming all the more crucial. The linkage of molecular and morphological approaches is worth more of the attention of researchers because this approach provides new knowledge for better understanding insufficiently investigated lethal diseases caused by haemosporidian infections, particularly on the exoerythrocytic (tissue) and vector stages. That requires close collaboration between researchers from different fields with a common interest.

Plasmodium spp.: An experimental study on vertebrate host susceptibility to avian malaria.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (<0.01%) and transient in all exposed birds. This study provides novel information about susceptibility of avian hosts to a wide array of malaria parasite lineages, outlining directions for future experimental research on various aspects of biology and epidemiology of avian malaria.

Primary peak and chronic malaria infection levels are correlated in experimentally infected great reed warblers.

Malaria parasites often manage to maintain an infection for several months or years in their vertebrate hosts. In humans, rodents and birds, most of the fitness costs associated with malaria infections are in the short initial primary (high parasitaemia) phase of the infection, whereas the chronic phase (low parasitaemia) is more benign to the host. In wild birds, malaria parasites have mainly been studied during the chronic phase of the infection. This is because the initial primary phase of infection is short in duration and infected birds with severe disease symptoms tend to hide in sheltered places and are thus rarely caught and sampled. We therefore wanted to investigate the relationship between the parasitaemia during the primary and chronic phases of the infection using an experimental infection approach. We found a significant positive correlation between parasitaemia in the primary peak and the subsequent chronic phase of infection when we experimentally infected great reed warblers (Acrocephalus arundinaceus) with Plasmodium ashfordi. The reason for this association remains to be understood, but might arise from individual variation in exoerythrocytic parasite reservoirs in hosts, parasite antigenic diversity and/or host genetics. Our results suggest that the chronic phase parasitaemia can be used to qualitatively infer the parasitaemia of the preceding and more severe primary phase, which is a very important finding for studies of avian malaria in wild populations.

Contrasting Patterns of Genetic Differentiation among Blackcaps (Sylvia atricapilla) with Divergent Migratory Orientations in Europe

Migratory divides are thought to facilitate behavioral, ecological, and genetic divergence among populations with different migratory routes. However, it is currently contentious how much genetic divergence is needed to maintain distinct migratory behavior across migratory divides. Here we investigate patterns of neutral genetic differentiation among Blackcap (Sylvia atricapilla) populations with different migratory strategies across Europe. We compare the level of genetic divergence of populations migrating to southwestern (SW) or southeastern (SE) wintering areas with birds wintering in the British Isles following a recently established northwesterly (NW) migration route. The migratory divide between SW and SE wintering areas can be interpreted as a result of a re-colonization process after the last glaciation. Thus we predicted greater levels of genetic differentiation among the SW/SE populations. However, a lack of genetic differentiation was found between SW and SE populations, suggesting that interbreeding likely occurs among Blackcaps with different migratory orientations across a large area; therefore the SW/SE migratory divide can be seen as diffuse, broad band and is, at best, a weak isolating barrier. Conversely, weak, albeit significant genetic differentiation was evident between NW and SW migrants breeding sympatrically in southern Germany, suggesting a stronger isolating mechanism may be acting in this population. Populations located within/near the SW/SE contact zone were the least genetically divergent from NW migrants, confirming NW migrants likely originated from within the contact zone. Significant isolation-by-distance was found among eastern Blackcap populations (i.e. SE migrants), but not among western populations (i.e. NW and SW migrants), revealing different patterns of genetic divergence among Blackcap populations in Europe. We discuss possible explanations for the genetic structure of European Blackcaps and how gene flow influences the persistence of divergent migratory behaviors.

Spatial and temporal differences in the blood parasite fauna of passerine birds during the spring migration in Bulgaria

Species composition and prevalence of the blood parasites of three migratory Acrocephalus species of warblers (Acrocephalus arundinaceus, Acrocephalus scirpaceus and Acrocephalus schoenobaenus) were studied during spring period in Bulgaria. For each of the studied species, we compared the parasite fauna during their spring migration at three study sites in search of infection patterns in time and space. Spatial differences were registered in the blood parasites and their prevalence for two of the studied species—A. arundinaceus and A. scirpaceus, whereas for the third one—A. schoenobaenus, there were no significant differences. The parasite species Haemoproteus belopolskyi was present in migrating A. arundinaceus only at Kalimok station, but not at the other two sampling locations. Plasmodium prevalence in A. arundinaceus migrating through Kalimok was also different between years. A decrease of H. belopolskyi prevalence was detected for A. schoenobaenus at Petarch during the spring of 2006. On the contrary, Plasmodium (Haemamoeba) sp. infections of the same bird species increased during the spring period. The differences found between sites could be explained with the different origin of the migrants at different migration stopover points. Climatic conditions and vector abundance in the zones of origin are probable reasons for the observed differences between years.

Two new species of Haemoproteus Kruse, 1890 (Haemosporida, Haemoproteidae) from European birds, with emphasis on DNA barcoding for detection of haemosporidians in wildlife

Two new species of Haemoproteus Kruse, 1890 (Haemosporida, Haemoproteidae) are described: Haemoproteus (Parahaemoproteus) homovelans n. sp. from Grey-faced Woodpecker, Picus canus Gmelin, and Haemoproteus (Parahaemoproteus) concavocentralis n. sp. recorded in Hawfinch, Coccothraustes coccothraustes (Linnaeus), both sampled in Bulgaria. The morphology of the gametocytes and their host-cells are described and mitochondrial cytochrome b (cyt b) gene sequences are generated. Haemoproteus homovelans possesses circumnuclear gametocytes lacking volutin granules. This parasite is particularly similar to Haemoproteus velans Coatney & Roudabush, 1937 also possessing circumnuclear gametocytes that are, however, overfilled with volutin. Haemoproteus concavocentralis can be readily distinguished from all described avian haemoproteids due to the presence of an unfilled concave space between the central part of advanced gametocytes and erythrocyte nucleus. Bayesian phylogenetic analyses of 40 haemosporidian cyt b lineages showed close relationships of H. concavocentralis (hHAWF2) with a group of Haemoproteus spp. possessing gametocytes that are pale-stained with Giemsa. The lineage hPICAN02 of H. homovelans clustered with parasites infecting non-passerine birds. Phylogenetic analyses support the current subgeneric classification of the avian haemoproteids and suggest that cyt b lineage hPIPUB01 (GenBank EU254552) has been incorrectly assigned to Haemoproteus picae Coatney & Roudabush, 1937, a common parasite of corvid birds (Passeriformes). This study emphasises the importance of combining molecular techniques and light microscopy in the identification and field studies of avian haemosporidian parasites. Future development of barcodes for molecular identification of haemoproteids will allow better diagnostics of these infections, particularly in veterinary studies addressing insufficiently investigated tissue pathology caused by these parasites.

Host preferences of ornithophilic biting midges of the genus Culicoides in the Eastern Balkans.

Many biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of a diverse number of pathogens. The identification of their feeding behaviour and of vector–host associations is essential for understanding their transmission capacity. By applying two different nested polymerase chain reaction (PCR) assays, of which one targeted the avian cyt b gene and the other targeted the COI gene of a wide range of vertebrates, we identified the blood hosts of six biting midge species including Culicoides circumscriptus, Culicoides festivipennis, Culicoides punctatus, Culicoides pictipennis, Culicoides alazanicus and Culicoides cf. griseidorsum, the latter two of which are reported in Bulgaria for the first time. Bird DNA was found in 50.6% of 95 investigated bloodmeals, whereas mammalian DNA was identified in 13.7%. Two Culicoides species were found to feed on both birds and mammals. There was remarkable diversity in the range of avian hosts: 23 species from four orders were identified in the abdomens of four Culicoides species. The most common bird species identified was the magpie, Pica pica (n = 7), which was registered in all four ornithophilic biting midge species. Six bloodmeals from the great tit, Parus major, were recorded only in C. alazanicus. None of the studied species of Culicoides appeared to be restricted to a single avian host.

Timing avian long-distance migration: from internal clock mechanisms to global flights

Migratory birds regularly perform impressive long-distance flights, which are timed relative to the anticipated environmental resources at destination areas that can be several thousand kilometres away. Timely migration requires diverse strategies and adaptations that involve an intricate interplay between internal clock mechanisms and environmental conditions across the annual cycle. Here we review what challenges birds face during long migrations to keep track of time as they exploit geographically distant resources that may vary in availability and predictability, and summarize the clock mechanisms that enable them to succeed. We examine the following challenges: departing in time for spring and autumn migration, in anticipation of future environmental conditions; using clocks on the move, for example for orientation, navigation and stopover; strategies of adhering to, or adjusting, the time programme while fitting their activities into an annual cycle; and keeping pace with a world of rapidly changing environments. We then elaborate these themes by case studies representing long-distance migrating birds with different annual movement patterns and associated adaptations of their circannual programmes. We discuss the current knowledge on how endogenous migration programmes interact with external information across the annual cycle, how components of annual cycle programmes encode topography and range expansions, and how fitness may be affected when mismatches between timing and environmental conditions occur. Lastly, we outline open questions and propose future research directions. This article is part of the themed issue ‘Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals’.