Location and timing of infection drives a sex-bias in Haemoproteus prevalence in a hole-nesting bird.

Sex biases in prevalence of disease are often attributed to intrinsic factors, such as physiological differences while a proximate role of extrinsic factors such as behavioural or ecological differences may be more difficult to establish. We combined large-scale screening for the presence and lineage identity of avian malaria (haemosporidian) parasites, in 1234 collared flycatchers (Ficedula albicollis) with life-history information from each bird to establish the location and timing of infection. We found an overall infection rate of 36.2% ± 0.03 (95% CI) with 25 distinct malaria lineages. Interestingly, first-year breeding males and females had similar infection prevalence while females accrued a significantly higher infection rate than males later in life. The sex difference in infection rate was driven by the most abundant Haemoproteus, lineage, hPHSIB1, while the infection rate of Plasmodium lineages was similar in males and females. Furthermore, when infections were assigned to an apparent transmission location, we found that the sex difference in infection rate trend was driven by lineages transmitted in Europe, more specifically by one lineage (the hPHSIB1), while no similar pattern was found in African lineages. We deduce that the observed infection patterns are likely to be caused by differences in breeding behaviour, with incubating females (and nestling individuals of both sexes) being easy targets for the biting insects that are the vectors of avian malaria parasites. Overall, our results are most consistent with ecological factors rather than intrinsic factors underlying the observed sex-biased infection rate of avian malaria in collared flycatchers.

First record and molecular diversity of haemosporidian parasites in Sula leucogaster (Boddaert, 1783) from the southeastern Brazilian coast.

Avian haemosporidian parasites are protozoans transmitted by vectors, belonging to the genera Plasmodium, Haemoproteus, and Leucocytozoon, capable of infecting various bird species. The prevalence of these parasites varies depending on factors such as the habitat and geographical location of the host and vector, as well as across different bird groups. Seabirds typically exhibit low infection rates due to the absence of vectors in marine environments. This study aimed to identify and characterize the molecular diversity of Haemosporida parasites in a population of Brown Boobies (Sula leucogaster) (n = 37) from the Santana Archipelago in Macaé, Rio de Janeiro state. We utilized PCR-based detection methods to confirm the presence of haemosporidian parasites and sequencing (ctyb gene) to delineate the lineages. The results uncovered a notable prevalence (48.6%) of haemosporidian parasites (Plasmodium and Haemoproteus), comprising 12 novel lineages and four previously documented lineages (establishing new host records). Our findings suggest that transmission and infection by these hemoparasites can occur both on and off the island. Overall, this study yields valuable insights into the genetic diversity of haemosporidian parasites in Brown Boobies, enriching our understanding of the ecology and epidemiology of these parasites in seabirds.

AVIAN HAEMOSPORIDIANS IN GREATER SCAUP (AYTHYA MARILA) AND LESSER SCAUP (AYTHYA AFFINIS) FROM WISCONSIN.

Avian haemosporidians are a diverse group of protozoan parasites that infect a wide range of host species. Waterfowl are an ecologically and economically important group of hosts that have been underrepresented in studies of haemosporidians. Diving ducks have unique life history traits, and morphological, behavioral, and dietary differences separate them from more common dabbling ducks. Greater scaup (Aythya marila) and lesser scaup (Aythya affinis) are closely related diving ducks with declining population trends in North America. To better understand the diversity of haemosporidians within diving ducks and factors related to host infections in scaup, we surveyed 82 hunter-donated waterfowl from 8 species of divers, sea ducks, and dabblers from Green Bay, Wisconsin from 2019 to 2021. We used molecular detection methods and phylogenetic and statistical analyses to describe the diversity, host associations, and prevalence of haemosporidians. We detected 14 unique genetic lineages of haemosporidians, including 4 novel lineages. We identified at least 1 lineage of haemosporidian in each of the 8 host species of divers, sea ducks, and dabblers examined. Lesser scaup had more diverse haemosporidian communities than did greater scaup, but lineages showed no clustering among these hosts when incorporated in phylogenetic analyses with lineages from other Nearctic waterfowl. Female lesser scaup had the highest infection prevalence, but there was no effect of host age or year of sampling. Our findings underscore the importance of species and sex differences that could lead to a higher risk of infections. Our results also fill an important geographical sampling gap for haemosporidians along a key migratory route. Increased monitoring of haemosporidians in waterfowl could contribute to insights into parasite evolution and ecology and the conservation and management of host populations.

The Impact of Avian Haemosporidian Infection on Feather Quality and Feather Growth Rate of Migratory Passerines.

Bird feathers have several functions, including flight, insulation, communication, and camouflage. Since feathers degrade over time, birds need to moult regularly to maintain these functions. However, environmental factors like food scarcity, stress, and parasite infections can affect feather quality and moult speed. This study examined the impact of avian haemosporidian infection and uropygial gland volume, as well as feather quality and feather growth rate in two migratory hirundine species captured in southwestern Spain-the house martin (Delichon urbicum) and sand martin (Riparia riparia). Our findings showed that the prevalence of infection varied among species, with house martins having the highest rates, possibly due to their larger colony size. Moreover, haemosporidian infection had a different impact on each species; infected house martins exhibited lower feather quality than healthy individuals, although this outcome was not observed in sand martins. Furthermore, no effect of infection on feather growth rate was observed in both hirundinids. Additionally, feather growth rate only correlated positively with feather quality in house martins. Finally, no link was observed between uropygial gland volume and feather quality or feather growth rate in any of the species in this study. These findings highlight the effect of haemosporidian infections on the plumage of migratory birds, marking, for the first time, how avian haemosporidian infection is shown to adversely impact feather quality. Even so, further research is needed to explore these relationships more deeply.

A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies.

BACKGROUND: Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. METHODS: A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. RESULTS: The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. CONCLUSIONS: This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

Avian Haemosporidian Infection in Wildlife Rehabilitation Centres of Portugal: Causes, Consequences, and Genetic Diversity.

In the last decade, over 40% of bird species in Europe have experienced poor and bad conservation status, with more than 30% of bird species in mainland Portugal threatened with extinction. Along with anthropogenic factors, parasites and pathogens such as avian haemosporidians have been suggested to be responsible for these avian population declines. Wildlife rehabilitation centres play an essential role in species conservation and preservation. Moreover, animals admitted for rehabilitation can provide valuable information regarding transmission and pathogenicity of many diseases that affect wild birds that are rarely sampled in nature. However, reports of haemosporidians in captive birds are still limited. Here, we explored the prevalence and genetic diversity of avian haemosporidians in 89 birds from 29 species admitted to rehabilitation centres in Portugal, showing an overall infection prevalence of 30.3%. The prevalence of infection was higher in Strigiformes and in birds admitted to rehabilitation centres due to debilitating diseases. Remarkably, 30% of the infected bird species have not been found to harbour malaria parasites in preceding studies. We detected 15 different haemosporidian lineages infecting a third of bird species sampled. Notably, 2 out of these 15 detected haemosporidian lineages have not been obtained previously in other studies. Furthermore, we also identified nine new host-parasite interactions representing new host records for these haemosporidian parasites. Finally, our results revealed that birds infected with haemosporidians require longer rehabilitation treatments, which increase the economic costs for rehabilitation and may impair their survival prospects. These findings emphasise the importance of integrating haemosporidian infection considerations into rehabilitation protocols, highlighting the challenges posed by these infections in avian conservation and rehabilitation, including economic and logistical demands.

Molecular survey and genetic diversity of Plasmodium sp. infesting domestic poultry in northeastern Thailand.

Introduction: Haemosporidian parasites are prevalent worldwide and can cause economic losses in poultry production. These parasites are arousing interest in Thailand and are found in many avian species. There is insufficient information on the genetic diversity of these alveolates from the largest families - Plasmodidae, Haemoprotidae and Leucocytozoidae - specifically parasitising ducks, turkeys, and geese. Material and Methods: Blood samples from 116 backyard poultry (60 ducks, 36 turkeys and 20 geese) in northeastern Thailand were investigated for Plasmodium spp., Haemoproteus spp. and Leucocytozoon spp. infections using microscopic examination and molecular approaches. Results: A total of 37/116 birds (31.9%) had confirmed Plasmodium infections. The prevalence was 69.4% (25/36) in turkeys, 18.3% (11/60) in ducks, and 5.0% (1/20) in geese. Of these 37 positives, 86.5% were Plasmodium sp., 10.8% were P. gallinaceum and 2.7% were P. juxtanucleare. Sequence analysis based on the cytochrome b gene identified seven lineages, of which two were new lineages in backyard poultry. Conclusion: This is the first report on the prevalence of haemosporidian parasites in backyard poultry in northeastern Thailand. The results provide important data for better understanding the molecular epidemiology of haemosporidian parasites infection in poultry in this region, which will be helpful in controlling these blood parasites.

High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

Brain parasites and misorientation of migratory birds.

Haemoproteus blood parasites of birds are thought to be relatively benign. Recent findings show that infections may develop in the brain of birds, possibly distorting their orientation sense. Misdirected migration may lead migrants outside their range where they are recognized as vagrants and can transmit parasites to novel hosts.

Differences in haemosporidian parasite prevalence and diversity in migratory and resident birds of prey species revealed by a non-invasive sampling method.

Avian haemosporidian parasites are widespread globally and infect numerous wild bird species. However, they have primarily been studied in passerine birds. Accordingly, the prevalence and diversity of these parasites in birds of prey remain understudied. This lack of research is primarily due to the international protection status of many of these species, their sparse distribution across ecosystems and difficulty to capture in the wild. The aim of this study was to examine the prevalence and diversity of haemosporidian parasites in two species of birds of prey, namely white-tailed eagle and lesser spotted eagle. To achieve this, a non-invasive approach was employed, involving the extraction of DNA from blood spots present in moulted feathers. Freshly moulted feathers were collected from the ground under nests or within the nests of these birds during the breeding season. A visible blood spot located at the junction of the calamus and rachis was removed and fixed in SET buffer for molecular analysis. The identification of haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) was conducted using PCR-based methods. Overall, parasite DNA was successfully detected in shed feathers that were kept in their original form at least decade prior to analysis. Among the studied individuals, resident white-tailed eagles showed significantly lower infection rates of haemosporidian parasites compared to migratory lesser spotted eagles. A total of nine genetic lineages of haemosporidian parasites were detected, with seven of them being new to science based on partial sequences of the cytb gene. Additionally, the phylogenetic relationships among these new lineages and previously described ones were established. These findings highlight the suitability of non-invasive sampling for investigating the prevalence and diversity of haemosporidian parasites in wild birds of prey populations. Moreover, this approach holds promise for studying other challenging-to-reach and protected bird species. According to our research, there is a greater chance of finding haemosporidian parasites in freshly gathered feathers.

Therapeutic management of pseudomalaria in a flock of pigeons with chloroquine.

Two Indian rock pigeons aged 2-3 months presented to the Referral Veterinary Polyclinic and Teaching Veterinary Clinical Complex, Indian Veterinary Research Institute, Utter Pradesh with a history of decreased feed intake, twisting of the neck, and inability to fly. The same symptoms also caused the deaths of two other birds from the same flock. The bird seemed dull and depressed during a clinical examination, had ruffled feathers, a tilted head and circling. Examination of a faecal sample showed no intestinal parasites. Upon observation of a blood smear, many intracytoplasmic characteristic halter-shaped Hemoproteus columbae gametocytes could be detected. The case was diagnosed as pigeon pseudomalaria. The treatment was initiated with chloroquine@10 mg/kg body weight in drinking water for 5 days along with the multivitamin supplementation for one week. Permethrin spray was applied externally to the whole flock in the house to get rid of the fly vector. The clinical state of the birds was evaluated one week after initiation of the therapy. The pigeon had an uneventful recovery and the blood smear examination revealed no haemoparasites.

Molecular characterization of Haemoproteus enucleator with emphasis on the host and geographic distribution.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan and highly diverse blood parasites of birds that have been neglected in avian medicine. However, recent discoveries based on molecular diagnostic markers show that these pathogens often cause marked damage to various internal organs due to exo-erythrocytic development, sometimes resulting in severe and even lethal avian haemoproteosis, including cerebral pathologies. Molecular markers are essential for haemoproteosis diagnostics, but the data is limited, particularly for parasites transmitted in tropical ecosystems. This study combined microscopic and molecular approaches to characterize Haemoproteus enucleator morphologically and molecularly. Blood samples were collected from the African pygmy kingfisher Ispidina picta in Cameroon, and the parasite was identified using morphological characters of gametocytes. The analysis of partial cytochrome b sequences (cytb) identified a new Haemoproteus lineage (hISPIC03), which was linked to the morphospecies H. enucleator. Illustrations of blood stages were provided and the phylogenetic analysis showed that the new lineage clustered with five other closely related lineages belonging to the same morphospecies (hALCLEU01, hALCLEU02, hALCLEU03, hISPIC01, and hALCQUA01), with a maximum genetic distance between these lineages of 1.5 % (7 bp difference) in the 478 bp cytb sequences. DNA haplotype network was developed and identified geographic and host distribution of all lineages belonging to H. enucleator group. These lineages were almost exclusively detected in African kingfishers from Gabon, Cameroon, South Africa, and Botswana. This study developed the molecular characterization of H. enucleator and provides opportunities for diagnostics of this pathogen at all stages of its life cycle, which remains undescribed in all its closely related lineages.

Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species.

Avian haemosporidian parasites (order Haemosporida, phylum Apicomplexa) are blood and tissue parasites transmitted by blood-sucking dipteran insects. Three genera (Plasmodium, Haemoproteus and Leucocytozoon) have been most often found in birds, with over 270 species described and named in avian hosts based mainly on the morphological characters of blood stages. A broad diversity of Haemoproteus parasites remains to be identified and characterized morphologically and molecularly, especially those infecting birds of prey, an underrepresented bird group in haemosporidian parasite studies. The aim of this study was to investigate and identify Haemoproteus parasites from a large sample comprising accipitriform raptors of 16 species combining morphological and new molecular protocols targeting the cytb genes of this parasite group. This study provides morphological descriptions and molecular characterizations of two Haemoproteus species, H. multivacuolatus n. sp. and H. nisi Peirce and Marquiss, 1983. Haemoproteus parasites of this group were so far found in accipitriform raptors only and might be classified into a separate subgenus or even genus. Cytb sequences of these parasites diverge by more than 15% from those of all others known avian haemosporidian genera and form a unique phylogenetic clade. This study underlines the importance of developing new diagnostic tools to detect molecularly highly divergent parasites that might be undetectable by commonly used conventional tools.

Title: Nouveau clade phylogénétique de parasites de rapaces Accipitridae du genre Haemoproteus (Haemosporida, Haemoproteidae), avec description d'une nouvelle espèce d'Haemoproteus. Abstract: Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d'oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d'étudier et d'identifier les Haemoproteus à partir d'un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d'Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n'ont jusqu'à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d'hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l'importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés.

Molecular Survey of Haemosporidian Parasites in Procellariiformes Sampled in Southern Brazil, 2013-22.

The order Procellariiformes includes several species of seabirds that perform long-distance migrations crossing all the oceans. These movements may contribute to the dispersal and exchange of hemoparasites, such as haemosporidians. There is a lack of studies regarding the order Haemosporida in Procellariiformes, and, to date, only the genus Plasmodium has been reported. This survey investigated the occurrence of the three genera of haemosporidians, Plasmodium, Haemoproteus, and Leucocytozoon, in samples collected between 2013 and 2022 from 95 individuals of 14 species of Procellariiformes from southern Brazil, including live animals in rehabilitation centers, individuals caught as incidental bycatch, and carcasses found along the coast. A total of 171 samples of blood and fragments of liver and spleen were analyzed, with extracted DNA being subjected to a nested PCR followed by phylogeny analysis. All animals were negative for Plasmodium spp. and Leucocytozoon spp., but one Black-browed Albatross (Thalassarche melanophris) and one Manx Shearwater (Puffinus puffinus) specimen were positive for Haemoproteus spp. The sequences obtained from positive seabirds did not show 100% similarity with other known lineages available in the MalAvi database and thus were probably novel lineages. However, one sequence clustered together with Haemoproteus noctuae, a parasite from Strigiformes, while the other was grouped with Haemoproteus columbae, which is classically related to Columbiformes. These results suggest that both positive animals may have become infected when beached or in rehabilitation centers by a spillover of vectors from local birds. This highlights the importance of surveillance of the health of Procellariiformes regarding the possibility of dissemination of new pathogens in different bird populations.

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.

Seasonal Variation in Detection of Haemosporidia in a Bird Community: A Comparison of Nested PCR and Microscopy.

In a 2-yr study on prevalence of Haemosporidia in an avian community in Ithaca, New York, USA, we tested the hypothesis that apparent seasonal variation in prevalence is influenced by the detection protocol. We confirmed a higher detection of Haemosporidia using a molecular diagnosis technique (PCR) than by microscopy; this further increased when the PCR test was triplicated. Microscopic examination and PCR techniques have different specificity and sensitivity and therefore different probabilities of detecting hemoparasites. Birds with chronic infections or sampled during winter often have very low parasitemia, and such infections may be missed by microscopy but detected by PCR. Haemosporidian prevalence was higher during the breeding season than during the nonbreeding season regardless of the method used. Detection of Leucocytozoon spp. infection from blood smears using microscopy was challenging.

The association between haemosporidian infection and non-breeding moult location in great reed warblers revisited by combining feather stable isotope profiles and geolocator data.

Stable isotope analysis provides valuable insights into the ecology of long-distance migratory birds during periods spent away from a specific study site. In a previous study, Swedish great reed warblers (Acrocephalus arundinaceus) infected with haemosporidian parasites differed in feather isotope ratios compared to non-infected birds, suggesting that infected and non-infected birds spent the non-breeding season in different locations or habitats. Here, we use a novel dataset comprising geolocator data, isotopes, and haemosporidian infection status of 92 individuals from four Eurasian populations to investigate whether parasite transmission varies with geography or habitats. We found that the probability of harbouring Plasmodium and Leucocytozoon parasites was higher in birds moulting in the eastern region of the non-breeding grounds. However, no geographic pattern occurred for Haemoproteus infections or overall infection status. In contrast to the previous study, we did not find any relationship between feather isotope ratios and overall haemosporidian infection for the entire current dataset. Plasmodium-infected birds had lower feather δ15N values indicating that they occupied more mesic habitats. Leucocytozoon-infected birds had higher feather δ34S values suggesting more coastal sites or wetlands with anoxic sulphate reduction. As the composition and prevalence of haemosporidian parasites differed between the old and the current dataset, we suggest that the differences might be a consequence of temporal dynamics of haemosporidian parasites. Our results emphasize the importance of replicating studies conducted on a single population over a restricted time period, as the patterns can become more complex for data from wider geographical areas and different time periods.

Haemosporidian parasite infections of Malagasy Philepittidae and Nectariniidae are driven by phylogeny rather than ecology.

The nectarivorous common sunbird asity (Neodrepanis coruscans) is phylogenetically closely related to the frugivorous velvet asity (Philepitta castanea), yet it shares similar habitat and foraging behaviour as the Malagasy sunbirds (Cinnyris spp.). As ecological factors have been shown to influence blood parasite prevalence, it should be tested whether parasite abundance, prevalence and diversity of N. coruscans are more similar to the sunbirds than to its relative. Therefore, blood samples (n = 156) and smears (n = 60) were tested for different blood parasites (Haemosporida, trypanosomes, filarioid nematodes) using molecular and microscopic methods. High prevalence of haemosporidian parasites was observed in all bird taxa, with rates ranging from 23% in N. coruscans to 84.6% in C. notatus. The Malagasy Cinnyris spp. exhibited a high occurrence of mixed haemosporidian infections (>76%) with various specialized lineages. Within the Philepittidae family, no Haemoproteus infection was detected and just a few cases of mixed infections. Nectariniidae species predominantly had specialized haemosporidian lineages, while Philepittidae had infections mainly caused by generalist lineages. These findings emphasize the diverse range of blood parasites in Nectariniidae, while additionally highlighting the high diversity of trypanosomes and filarioid nematodes in Philepittidae. Additionally, several newly discovered haemosporidian lineages, Trypanosoma isolates and filarioid nematode isolates were identified. Notably, Philepittidae exhibited a lower prevalence of avian haemosporidian parasites compared to Nectariniidae, possibly due to potential resistance mechanisms. Despite N. coruscans sharing similar habitat and behavioural ecology with both Cinnyris spp., it closely resembles its relative, P. castanea, in all aspects of haemosporidian parasitism.

Contribution to ecological environmental factors and the occurrence of haemosporidians in birds in Zhongar Alatau National Park, Kazakhstan.

In addition to the presence of a suitable host and vector, the prevalence of haemosporidians is influenced by several important factors, including the environmental conditions of the habitat, which depend on broader geographic characteristics. The aim of this study is to perform a preliminarily assessment of the distribution of blood parasites in birds from the mountainous area of Zhongar Alatau NP and to find potential new sites for research on their ecology in Kazakhstan. The results of this research constitute the first report on the occurrence of blood parasites from this area. A total of 58 birds, from the order Passeriformes and one individual from the order Caprimulgiformes, were examined during the study. The overall prevalence of infections caused by haemosporidian parasites (Haemoproteus, Leucocytozoon) was 18.6%. Neither the genus Plasmodium nor the presence of trypanosomes and microfilariae was detected in the birds examined. Three birds (5.1% prevalence) were infected with parasites of the genus Haemoproteus, in all eleven positive birds the analyses showed the presence of parasites of the genus Leucocytozoon (18.6% prevalence). The presence of parasites genus Haemoproteus was detected only in birds that were also infected with Leucocytozoon parasites. More infections with parasites of the genus Leucocytozoon are predicted due to the higher altitude and ecological factors at the capture sites, which are more favourable for the development of vectors of this genus. The species Haemoproteus majoris was detected in the host Emberiza cioides and species Haemoproteus minutus in host Turdus merula. Other species of this genus in the hosts Cyanistes cyanus and Turdus atrogularis were not determined. The species Leucocytozoon fringilinarum was detected in the hosts Cyanistes cyanus and Parus major, Leucocytozoon dubreuili was detected in Turdus atrogularis and Turdus merula. In the other host species Aegithalos caudatus, Emberiza cioides and Periparus aterus, it was not possible to dermine the species of the genus Leucocytozoon.