Leucocytozoon cariamae n. sp. and Haemoproteus pulcher coinfection in Cariama cristata (Aves: Cariamiformes): first mitochondrial genome analysis and morphological description of a leucocytozoid in Brazil.

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L. fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus. We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

Avian haemosporidians of breeding birds in the Davis Mountains sky-islands of west Texas, USA.

Avian haemosporidians are protozoan parasites transmitted by insect vectors that infect birds worldwide, negatively impacting avian fitness and survival. However, the majority of haemosporidian diversity remains undescribed. Quantifying this diversity is critical to determining parasite­host relationships and host-switching potentials of parasite lineages as climate change induces both host and vector range shifts. In this study, we conducted a community survey of avian haemosporidians found in breeding birds on the Davis Mountains sky islands in west Texas, USA. We determined parasite abundance and host associations and compared our results to data from nearby regions. A total of 265 birds were screened and infections were detected in 108 birds (40.8%). Most positive infections were identified as Haemoproteus (36.2%), followed by Plasmodium (6.8%) and Leucocytozoon (0.8%). A total of 71 haemosporidian lineages were detected of which 39 were previously undescribed. We found that regional similarity influenced shared lineages, as a higher number of lineages were shared with avian communities in the sky islands of New Mexico compared to south Texas, the Texas Gulf Coast and central Mexico. We found that migratory status of avian host did not influence parasite prevalence, but that host phylogeny is likely an important driver.

Assessing rates of parasite coinfection and spatiotemporal strain variation via metabarcoding: Insights for the conservation of European turtle doves Streptopelia turtur.

Understanding the frequency, spatiotemporal dynamics and impacts of parasite coinfections is fundamental to developing control measures and predicting disease impacts. The European turtle dove (Streptopelia turtur) is one of Europe's most threatened bird species. High prevalence of infection by the protozoan parasite Trichomonas gallinae has previously been identified, but the role of this and other coinfecting parasites in turtle dove declines remains unclear. Using a high-throughput sequencing approach, we identified seven strains of T. gallinae, including two novel strains, from ITS1/5.8S/ITS2 ribosomal sequences in turtle doves on breeding and wintering grounds, with further intrastrain variation and four novel subtypes revealed by the iron-hydrogenase gene. High spatiotemporal turnover was observed in T. gallinae strain composition, and infection was prevalent in all populations (89%-100%). Coinfection by multiple Trichomonas strains was rarer than expected (1% observed compared to 38.6% expected), suggesting either within-host competition, or high mortality of coinfected individuals. In contrast, coinfection by multiple haemosporidians was common (43%), as was coinfection by haemosporidians and T. gallinae (90%), with positive associations between strains of T. gallinae and Leucocytozoon suggesting a mechanism such as parasite-induced immune modulation. We found no evidence for negative associations between coinfections and host body condition. We suggest that longitudinal studies involving the recapture and investigation of infection status of individuals over their lifespan are crucial to understand the epidemiology of coinfections in natural populations.

Contrasting haemoparasite prevalence in larid species with divergent ecological niches and migration patterns.

Haemoparasites represent a diverse group of vector-borne parasites that infect a wide range of vertebrate hosts. In birds, haemoparasite infection rates may be associated with various ecological and life history traits, including habitat choice, colony size and migration distance. Here, we molecularly assessed the prevalence of 3 main haemoparasite genera (Plasmodium, Haemoproteus and Leucocytozoon) in 2 bird species with different habitat preferences and migratory behaviour: black-headed gulls (Chroicocephalus ridibundus) and common terns (Sterna hirundo). We found that gulls showed a much higher prevalence and diversity of Plasmodium or Haemoproteus (ca. 60% of individuals infected) than terns (zero prevalence). The prevalence of Leucocytozoon was low in both species (<3%). The differences in haemoparasite prevalences may be primarily driven by varying vector encounter rate resulting from different habitat preferences, as black-headed gulls mainly use vector-rich vegetated freshwater habitats, whereas common terns often use vector-poor coastal and brackish habitats. Since common terns migrate further than black-headed gulls, our results did not provide support for an association between haemoparasite prevalence and migratory distance. In gulls, we found a negative association between colony size and infection rates, suggestive of an ideal despotic distribution, and phylogenetic analyses of detected haemoparasite lineages provided evidence for higher host specificity in Haemoproteus than Plasmodium. Our results suggest that the preference for coastal areas and less vegetated habitats in terns may reduce haemoparasite infection rates compared to other larids, regardless of their migratory distance, emphasizing the role of ecological niches in parasite exposure.

Migratory behaviour does not alter cophylogenetic congruence between avian hosts and their haemosporidian parasites.

Parasites display various degrees of host specificity, reflecting different coevolutionary histories with their hosts. Avian hosts follow multiple migration patterns representing short but also long distances. As parasites infecting migratory birds are subjected to multiple environmental and biotic changes through their flyways, migration may disrupt or strengthen cophylogenetic congruence between hosts and parasites. On the one hand, parasites might adapt to a single migratory host, evolving to cope with the specific challenges associated with the multiple habitats occupied by the host. On the other, as migrants can introduce parasites into new habitats, higher rates of host switching could also disrupt cophylogenetic patterns. We analysed whether migratory behaviour shapes avian haemosporidian parasite­host cophylogenetic congruence by testing if contributions of host­parasite links to overall congruence differ among resident and short-, variable- and long-distance migrants globally and within South America only. On both scales, we found significant overall cophylogenetic congruence by testing whether overall congruence differed between haemosporidian lineages and bird species. However, we found no difference in contribution towards congruence among links involving resident vs migratory hosts in both models. Thus, migratory behaviour neither weakens nor strengthens bird­haemosporidian cophylogenetic congruence, suggesting that other avian host traits are more influential in generating phylogenetic congruence in this host­parasite system.

Prevalence and diversity of haemosporidians in a migratory high-elevation hummingbird in North America.

Hummingbirds (Trochilidae) are sensitive to environmental changes because of their extraordinary ecology, metabolism, and the highest red blood cell counts found in any vertebrate. These physiological attributes may render hummingbirds particularly susceptible to the effects of haemosporidian (blood parasite) infections. Much of the research on haemosporidians in hummingbirds has been conducted in South America; less is known about haemosporidian diversity and prevalence in North America. We sought to determine the prevalence and diversity of haemosporidians in a high-elevation species, the Broad-tailed Hummingbird (Selasphorus platycercus). Blood samples (N = 314) from 25 sites in Colorado and Wyoming were screened for haemosporidians using microscopy (n = 311) and PCR (n = 301). Both microscopy and sequencing diagnostic techniques detected haemosporidians in the same 5 hummingbirds, with an overall prevalence of 1.59%. Positive samples were sequenced at the cytochrome b gene and identified Haemoproteus archilochus and two Haemoproteus sp. not previously detected in North America. No parasites of the genera Plasmodium or Leucocytozoon were detected. Our study provides the first report of the prevalence and diversity of haemosporidians in Broad-tailed Hummingbirds in the Rocky Mountains.

Prevalence and diversity of avian haemosporidian parasites across islands of Milne Bay Province, Papua New Guinea.

The taxonomically diverse and relatively understudied avifauna of Papua New Guinea's (PNG) island archipelagos provide a unique ecological framework for studying haemosporidian parasite differentiation and geographic structure. We implemented molecular and phylogenetic analyses of partial mitochondrial DNA sequences to assess the host distribution of 3 genera of vector-transmitted avian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) across a range of islands off the southeastern tip of PNG. We identified 40 new lineages of haemosporidians, including five lineages belonging to Leucocytozoon, a genus not previously described in this region. Leucocytozoon infections were only observed on the larger, human-inhabited islands. Lineages belonging to Haemoproteus were diverse and had broad geographic distribution. Compared to the mainland, Haemoproteus parasites on the smaller, more distant islands had greater host specificity and lower infection prevalence. The black sunbird (Leptocoma aspasia), a commonly caught species, was shown to be a rare host for Haemoproteus spp. infections. Moreover, although birds of the genus Pitohui harbor a neurotoxin (homobatrachotoxin), they demonstrated an infection prevalence comparable to other bird species. The islands of PNG display heterogeneous patterns of haemosporidian diversity, distribution and host-specificity and serve as a valuable model system for studying host-parasite-vector interactions.

Avian blood parasite richness decreases with major histocompatibility complex class I loci number.

Major histocompatibility complex (MHC) genes are among the most polymorphic in the vertebrate genome. The high allele diversity is believed to be maintained primarily by sexual and pathogen-mediated balancing selection. The number of MHC loci also varies greatly across vertebrates, most notably across birds. MHC proteins play key roles in presenting antigens on the cell surface for recognition by T cells, with class I proteins specifically targeting intracellular pathogens. Here, we explore the hypothesis that MHC class I diversity (measured as loci number) coevolves with haemosporidian parasite burden of the host. Using data on 54 bird species, we demonstrate that high-MHC class I diversity is associated with significantly lower richness of Plasmodium, Haemoproteus as well as overall haemosporidian parasite lineages, the former thus indicating more efficient protection against intracellular pathogens. Nonetheless, the latter associations were only detected when MHC diversity was assessed using cloning and not 454 pyrosequencing-based studies, nor across all genotyping methods combined. Our results indicate that high-MHC class I diversity might play a key role in providing qualitative resistance against diverse haemosporidian parasites in birds, but further clarification is needed for the origin of contrasting results when using different genotyping methods for MHC loci quantification.

Haemosporidian taxonomic composition, network centrality and partner fidelity between resident and migratory avian hosts.

Migration can modify interaction dynamics between parasites and their hosts with migrant hosts able to disperse parasites and impact local community transmission. Thus, studying the relationships among migratory hosts and their parasites is fundamental to elucidate how migration shapes host-parasite interactions. Avian haemosporidians are some of the most prevalent and diverse group of wildlife parasites and are also widely studied as models in ecological and evolutionary research. Here, we contrast partner fidelity, network centrality and parasite taxonomic composition among resident and non-resident avian hosts using presence/absence data on haemosporidians parasitic in South American birds as study model. We ran multilevel Bayesian models to assess the role of migration in determining partner fidelity (i.e., normalized degree) and centrality (i.e., weighted closeness) in host-parasite networks of avian hosts and their respective haemosporidian parasites. In addition, to evaluate parasite taxonomic composition, we performed permutational multivariate analyses of variance to quantify dissimilarity in haemosporidian lineages infecting different host migratory categories. We observed similar partner fidelity and parasite taxonomic composition among resident and migratory hosts. Conversely, we demonstrate that migratory hosts play a more central role in host-parasite networks than residents. However, when evaluating partially and fully migratory hosts separately, we observed that only partially migratory species presented higher network centrality when compared to resident birds. Therefore, migration does not lead to differences in both partner fidelity and parasite taxonomic composition. However, migratory behavior is positively associated with network centrality, indicating migratory hosts play more important roles in shaping host-parasite interactions and influence local transmission.

Rediscovery of Plasmodium (Huffia) huffi (Apicomplexa, Haemosporida): a lost lineage from toucans.

Literature data on toucans haemosporidians are scarce and all reports come from investigations in Brazil. Muniz et al. (Rev Bras Malariol 3: 339-356, Muniz et al., Rev Bras Malariol 3:339-356, 1951) and Muniz and Soares (Rev Bras Malar 611-617, Muniz J, Soares R de RL (1954) Nota sôbre um parasita do gênero Plasmodium encontrado no Ramphastos toco Müller, 1776, "Tucano-Açu", e diferente do Plasmodium huffi: Plasmodium pinottii n. sp. Rev Bras Malar 611 - 617.) described two Plasmodium species, P. huffi and P. pinottii, in Ramphastos toco. Later, Manwell and Sessler (J Protozol 18: 570-574, Manwell and Sessler, Malaria Parasites of Toucans J Protozol 18:570-574, 1971) established a new subspecies, P. nucleophilum toucani. In the last review on avian haemosporidians, Valkiunas (Valkiunas, Avian malaria parasites and other haemosporidia, CRC Press, New York, 2005) highlighted that P. huffi was insufficiently characterized, considering it a lost lineage. Also, the original description of P. huffi was considered insufficiently clear, due to a possible co-infection of the toucan hosts with a Novyella-like species. Here, we redescribed the species Plasmodium (Huffia) huffi based on morphological and molecular data, which were found in two toucan species, Ramphastos toco and Pteroglossus aracari from Brazil. Morphological features of the specimens are markedly the same as the original description. In R. toco, we observe two individuals infected, one infected only with P. huffi and one co-infected with P. huffi and the Novyella-like species, as observed in the original description. Also, we observe one R. toco infected only with the Novyella-like species, identified by morphological and molecular data as Plasmodium (Novyella) nucleophilum nucleophilum. In this way, it was possible to redescribe Plasmodium huffi in detail, without the doubt characters observed in the original description. Moreover, by applying species delimitation algorithms to a large Plasmodium phylogeny, we were able to identify new possible hosts for P. huffi and extend its geographic distribution to include North America.

Spatiotemporal patterns of avian host-parasite interactions in the face of biogeographical range expansions.

Exploration of interactions between hosts and parasitic symbionts is important for our understanding of the temporal and spatial distribution of organisms. For example, host colonization of new geographical regions may alter levels of infections and parasite specificity, and even allow hosts to escape from co-evolved parasites, consequently shaping spatial distributions and community structure of both host and parasite. Here we investigate the effect of host colonization of new regions and the elevational distribution of host-parasite associations between birds and their vector-transmitted haemosporidian blood parasites in two geological and geographical settings: mountains of New Guinea and the Canary Islands. Our results demonstrate that bird communities in younger regions have significantly lower levels of parasitism compared to those of older regions. Furthermore, host-parasite network analyses demonstrate that blood parasites may respond differently after arriving to a new region, through adaptations that allow for either expanding (Canary Islands) or retaining (New Guinea) their host niches. The spatial prevalence patterns along elevational gradients differed in the two regions, suggesting that region-specific biotic (e.g., host community) and abiotic factors (e.g., temperature) govern prevalence patterns. Our findings suggest that the spatiotemporal range dynamics in host-parasite systems are driven by multiple factors, but that host and parasite community compositions and colonization histories are of particular importance.

Geographical and host species barriers differentially affect generalist and specialist parasite community structure in a tropical sky-island archipelago.

Understanding why some parasites emerge in novel host communities while others do not has broad implications for human and wildlife health. In the case of haemosporidian blood parasites, epidemic wild bird mortalities on oceanic islands have been linked to Plasmodium spp., but not genera like Haemoproteus. Indeed, Haemoproteus is absent from many oceanic islands. By contrast, birds on continental islands share long coevolutionary histories with both Plasmodium and Haemoproteus, and are thus ideal model systems to elucidate eco-evolutionary endpoints associated with these parasites in oceanic islands. Here, we examine eco-evolutionary dynamics of avian haemosporidian in the Shola sky-island archipelago of the Western Ghats, India. Our analyses reveal that compared to Plasmodium, Haemoproteus lineages were highly host-specific and diversified via co-speciation with their hosts. We show that community structure of host-generalist Plasmodium was primarily driven by geographical factors (e.g. biogeographic barriers), while that of host-specialist Haemoproteus was driven by host species barriers (e.g. phylogenetic distance). Consequently, a few host species can harbour a high diversity of Plasmodium lineages which, in turn, are capable of infecting multiple host species. These two mechanisms can act in concert to increase the risk of introduction, establishment, and emergence of novel Plasmodium lineages in island systems.

Micro- and macroparasite species richness in birds: The role of host life history and ecology.

Identifying the factors shaping variation in parasite diversity among host species is crucial to understand wildlife diseases. Although micro- and macroparasites may exert different selective pressures on their hosts, studies investigating the determinants of parasite species richness in animals have rarely considered this divide. Here, we investigated the role of host life history and ecology in explaining the species richness of helminths (macroparasites) and haemosporidians (microparasites) in birds world-wide. We collated data from multiple global datasets on diverse bird traits (longevity, body mass, coloniality, migration distance/tendency, geographic range size and dietary and habitat breadths) and the species richness of their helminth and haemosporidian parasites. We tested predictors of helminth and haemosporidian parasite richness using phylogenetic generalized linear mixed models in a Bayesian framework. We found that, after controlling for research effort and host phylogeny, the richness of helminths, but not of haemosporidians, increased with host longevity, range size, migration distance and dietary breadth. Overall, these correlates were also important across different helminth groups (acanthocephalans, cestodes, nematodes and trematodes), and two additional ones (body mass, coloniality) emerged as important for cestodes and acanthocephalans. We propose that long life spans may promote the diversity of helminth parasite assemblages over evolutionary time, thus resulting in richer helminth faunas. Similarly, longer-distance migrations, larger ranges and broader dietary breadths are likely to lead to greater encounter rates and the accumulation of trophically transmitted helminths. In contrast, vector-borne haemosporidians may be influenced more by factors related to vector ecology than by the host traits included in the analyses. The lack of strong associations between haemosporidian species richness and host characteristics emphasizes the need to find appropriate traits to model the distribution and diversity of parasites with different environmental preferences in order to anticipate disease emergence risks associated with global change.

Haemoparasites of the pied flycatcher: inter-population variation in the prevalence and community composition.

The prevalence and community composition of haemoparasites can substantially differ among avian host populations, which may lead to different selection pressures. Therefore, information about these parameters is crucial for understanding, e.g. the inter-population variation in host life history traits. Here, we molecularly screened a population of a long-distance migrant, the pied flycatcher Ficedula hypoleuca, from central Poland for the presence of three genera of blood parasites: Haemoproteus, Plasmodium and Trypanosoma. The infection rate in this population was the highest for haemosporidians (86·8%) and one of the highest for trypanosomes (39·7%) among the thus far screened breeding populations of this species. The haemosporidian community was composed of six Haemoproteus/Plasmodium lineages, and the trypanosome community - 4 species and a parasite assigned to genus level. Trypanosomes were dominated by T. culicavium, a recently described species, corroborating the prediction that insectivorous songbirds are vertebrate hosts of this parasite. Host sex and age did not explain variation in infection incidence except for the higher trypanosome infection rates in males. A comparison of the study population with three other breeding populations previously screened molecularly for haemosporidians showed some geographic differences. This study confirms the importance of examining local parasite communities across a host distribution range.

A new pathogen spillover from domestic to wild animals: Plasmodium juxtanucleare infects free-living passerines in Brazil.

Habitat modification may facilitate the emergence of novel pathogens, and the expansion of agricultural frontiers make domestic animals important sources of pathogen spillover to wild animals. We demonstrate for the first time that Plasmodium juxtanucleare, a widespread parasite from domestic chickens, naturally infects free-living passerines. We sampled 68 wild birds within and at the border of conservation units in central Brazil composed by Cerrado, a highly threatened biome. Seven out of 10 passerines captured in the limits of a protected area with a small farm were infected by P. juxtanucleare as was confirmed by sequencing a fragment of the parasite's cytochrome b. Blood smears from these positive passerines presented trophozoites, meronts and gametocytes compatible with P. juxtanucleare, meaning these birds are competent hosts for this parasite. After these intriguing results, we sampled 30 backyard chickens managed at the area where P. juxtanucleare-infected passerines were captured, revealing one chicken infected by the same parasite lineage. We sequenced the almost complete mitochondrial genome from all positive passerines, revealing that Brazilian and Asian parasites are closely related. P. juxtanucleare can be lethal to non-domestic hosts under captive and rehabilitation conditions, suggesting that this novel spillover may pose a real threat to wild birds.

Intra-individual changes in haemosporidian infections over the nesting period in great tit females.

Prevalence of haemosporidian parasites in bird populations varies temporally both between years and within a year. In contrast to variation at the population level, relatively little is known about variation in infection attributes at the individual level, especially in non-migratory species. We examined intra-individual changes in the presence and identity of haemosporidian parasites (genera Plasmodium and Haemoproteus) over the course of the nesting period in females of great tits (Parus major)-a species considered to be resident over much of its distribution range. Birds were sampled during two stages of the nesting period: nest building and nestling rearing. The mean time interval between sampling occasions was 43 days. Between the first and second samplings, 30.6% of females gained at least one parasite lineage and 18.5% lost the lineage. Haemoproteus gains were over three times more common than Plasmodium gains. The probability of the lineage gain decreased with the date of the first sampling, was higher in individuals in better body condition and differed between years, but was not associated with the host age. The probability of the lineage loss was not explained by any of the considered parameters except for year. These results indicate that in a large proportion of a population, infection attributes (presence/absence and/or parasite identity) may change over the nesting period and the occurrence of such changes may be associated with the individual quality. Consequently, this phenomenon should be taken into account to correctly interpret parasite-mediated effects.

Global warming will reshuffle the areas of high prevalence and richness of three genera of avian blood parasites.

The importance of parasitism for host populations depends on local parasite richness and prevalence: usually host individuals face higher infection risk in areas where parasites are most diverse, and host dispersal to or from these areas may have fitness consequences. Knowing how parasites are and will be distributed in space and time (in a context of global change) is thus crucial from both an ecological and a biological conservation perspective. Nevertheless, most research articles focus just on elaborating models of parasite distribution instead of parasite diversity. We produced distribution models of the areas where haemosporidian parasites are currently highly diverse (both at community and at within-host levels) and prevalent among Iberian populations of a model passerine host: the blackcap Sylvia atricapilla; and how these areas are expected to vary according to three scenarios of climate change. On the basis of these models, we analysed whether variation among populations in parasite richness or prevalence are expected to remain the same or change in the future, thereby reshuffling the geographic mosaic of host-parasite interactions as we observe it today. Our models predict a rearrangement of areas of high prevalence and richness of parasites in the future, with Haemoproteus and Leucocytozoon parasites (today the most diverse genera in blackcaps) losing areas of high diversity and Plasmodium parasites (the most virulent ones) gaining them. Likewise, the prevalence of multiple infections and parasite infracommunity richness would be reduced. Importantly, differences among populations in the prevalence and richness of parasites are expected to decrease in the future, creating a more homogeneous parasitic landscape. This predicts an altered geographic mosaic of host-parasite relationships, which will modify the interaction arena in which parasite virulence evolves.

Determinants of spring migration departure dates in a New World sparrow: Weather variables reign supreme.

Numerous factors influence the timing of spring migration in birds, yet the relative importance of intrinsic and extrinsic variables on migration initiation remains unclear. To test for interactions among weather, migration distance, parasitism, and physiology in determining spring departure date, we used the Dark-eyed Junco (Junco hyemalis) as a model migratory species known to harbor diverse and common haemosporidian parasites. Prior to spring migration departure from their wintering grounds in Indiana, USA, we quantified the intrinsic variables of fat, body condition (i.e., mass ~ tarsus residuals), physiological stress (i.e., ratio of heterophils to lymphocytes), cellular immunity (i.e., leukocyte composition and total count), migration distance (i.e., distance to the breeding grounds) using stable isotopes of hydrogen from feathers, and haemosporidian parasite intensity. We then attached nanotags to determine the timing of spring migration departure date using the Motus Wildlife Tracking System. We used additive Cox proportional hazard mixed models to test how risk of spring migratory departure was predicted by the combined intrinsic measures, along with meteorological predictors on the evening of departure (i.e., average wind speed and direction, relative humidity, and temperature). Model comparisons found that the best predictor of spring departure date was average nightly wind direction and a principal component combining relative humidity and temperature. Juncos were more likely to depart for spring migration on nights with largely southwestern winds and on warmer and drier evenings (relative to cooler and more humid evenings). Our results indicate that weather conditions at take-off are more critical to departure decisions than the measured physiological and parasitism variables.

Unravelling the patterns of exo-erythrocytic development of Haemoproteus parasites (Haemoproteidae, Haemosporida), with a case of abortive tissue stages in a naturally infected bird.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan blood parasites that affect bird fitness and health. Recent discoveries based on the application of molecular markers showed that exo-erythrocytic or tissue stages of haemoproteids damage various internal organs including the brain. However, the patterns of exo-erythrocytic development remain unclear for most of the described species. This study aimed to understand the exo-erythrocytic development of Haemoproteus parasitesin naturally infected Thrush nightingales Luscinia luscinia (Muscicapidae). Infections were confirmed in eight bird individuals by microscopic examination and PCR-based methods. Organs were examined using histology and in situ hybridization, which applied genus-specific and lineage-specific oligonucleotideprobes targeting the 18S rRNA of the parasites. Exo-erythrocytic meronts of Haemoproteus attenuatus (lineage hROBIN1) were found and described for the first known time in this avian host. Most meronts were seen in the lungs, with a few also present in the liver, heart, and pectoral muscle. The available data suggest that this parasite produces only meronts, and not megalomeronts. However, numerous megalomeronts at different stages of development were observed in the gizzard and the heart of one individual. Based on the morphology, location in organs, and diagnostics using the lineage-specific probes, the megalomeronts were attributed to Haemoproteu smajoris (lineage hWW2). Two cases of empty capsular-like walls of megalomeronts were seen in the gizzard, indicating that the megalomeronts had already ruptured and degenerated. The extensive microscopic examination did not reveal gametocytes of H. majoris, obviously indicating an abortive development. Abortive haemosporidian infections were often speculated to occur in wildlife but have not been documented in naturally infected birds. This study recognised patterns in the exo-erythrocytic development of H. attenuatus, and is to our knowledge the first documentation of abortive Haemoproteus infection in a naturally infected bird during exo-erythrocytic development.

Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae).

Avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs, a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6, Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.