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The drivers of regional parasite distributions are poorly understood, especially in comparison with those of free-living species. For vector-transmitted parasites, in particular, distributions might be influenced by host-switching and by parasite dispersal with primary hosts and vectors. We surveyed haemosporidian blood parasites (Plasmodium and Haemoproteus) of small land birds in eastern North America to characterize a regional parasite community. Distributions of parasite populations generally reflected distributions of their hosts across the region. However, when the interdependence between hosts and parasites was controlled statistically, local host assemblages were related to regional climatic gradients, but parasite assemblages were not. Moreover, because parasite assemblage similarity does not decrease with distance when controlling for host assemblages and climate, parasites evidently disperse readily within the distributions of their hosts. The degree of specialization on hosts varied in some parasite lineages over short periods and small geographic distances independently of the diversity of available hosts and potentially competing parasite lineages. Nonrandom spatial turnover was apparent in parasite lineages infecting one host species that was well-sampled within a single year across its range, plausibly reflecting localized adaptations of hosts and parasites. Overall, populations of avian hosts generally determine the geographic distributions of haemosporidian parasites. However, parasites are not dispersal-limited within their host distributions, and they may switch hosts readily.

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

Haemoproteus bobricklefsi sp. nov. (Haemosporida, Haemoproteidae) was found in the dunnock Prunella modularis and represents the first blood parasite described in accentor birds of the Prunellidae. The description is based on the morphology of blood stages and includes information about a barcoding segment of the mitochondrial cytochrome b gene (lineage hDUNNO01) and the full mitochondrial genome, which can be used for identification and diagnosis of this infection. The new parasite can be readily distinguished from described species of haemoproteids parasitizing passeriform birds due to markedly variable position of nuclei in advanced and fully grown macrogametocytes. Illustrations of blood stages of the new species are given, and phylogenetic analyses based on partial mitochondrial cytochrome b gene sequences and the full mitochondrial genome identified the closely related lineages. DNA haplotype networks showed that transmission occurs in Europe and North America. This parasite was found in the dunnock in Europe and several species of the Passerellidae in North America. It is probably of Holarctic distribution, with the highest reported prevalence in the UK. The parasite distribution seems to be geographically patchy, with preference for areas of relatively cool climates. Phylogenetic analysis suggests that H. bobricklefsi sp. nov. belongs to the Parahaemoproteus subgenus and is probably transmitted by biting midges belonging to Culicoides (Ceratopogonidae). The available data on molecular occurrence indicate that this pathogen is prone to abortive development, so worth attention in regard of consequences for bird health.

Background: The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods: Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results: Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. Conclusion: The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

Migratory animals likely play an important role in the geographic spread of parasites. In fact, a common assumption is that parasites are potentially transmitted by migratory animals at temporary stopover sites along migratory routes, yet very few studies have assessed whether transmission at stopover sites can or does occur. We investigated the potential for a group of vector-transmitted parasites, the avian haemosporidians, to be transmitted during migratory stopover periods at Rushton Woods Preserve in Pennsylvania, USA. Using an analysis of 1454 sampled avian hosts, we found that while a core group of abundant haemosporidians was shared between local breeding birds and passing migrants, the parasite community of migratory birds at Rushton was distinct from that of local breeding birds and showed similarity to a previously sampled boreal forest haemosporidian community. Haemosporidians that were unique to passing migratory birds were associated with sampling sites in North America with cooler summer temperatures than haemosporidians that are transmitted at Rushton, suggesting that the transmission of these parasites may be restricted to high-latitude regions outside of our temperate stopover site. We also found that the abundance of mosquitoes in our study region is offset from that of migratory bird abundance during avian migratory periods, with the peak period of bird migration occurring during periods of low mosquito activity. Collectively, these findings suggest that although abundant haemosporidians are possibly transmitted between local and passing migratory birds, a combination of biotic and abiotic factors may constrain haemosporidian transmission during avian stopover at our study site.

Blood parasites from the order Haemosporida infect many vertebrates and cause malaria-like diseases. In this study, a haemosporidian infection was detected in a sick grey crowned crane imported into China using a combination of morphological and molecular approaches. Blood samples were collected from the jugular vein and processed for morphological identification of infective parasites using stained blood smears and microscopy. No merogony occurs in the blood cells, and sporadic pigment granules were observed. Nested-PCR assays were employed for a molecular examination, which indicated that the cytb gene of this parasite had 94.1–94.9% identity to Haemoproteus antigonis . Subsequently, its mitochondrial genome structure was determined by high-throughput sequencing using the DNBSEQ-T7 platform. The determined structure was confirmed by the Sanger sequencing using amplicons. The mitochondrial genome obtained for this parasite exhibited a low CG content (32.0%) and possessed three protein-coding genes, encoding 1068 amino acids, which constituted 53.7% of the genome. Phylogenetic analysis indicated that this parasite clustered with Haemoproteus sp . is detected in grey crowned cranes from Africa. This parasite was likely acquired during importation of this animal; thus, strict quarantine of imported ornamental animals is required to prevent the entry of new pathogens.

Background: Haemosporidians (Haemosporida, Apicomplexa), which include malaria parasites, are found in nearly all terrestrial ecosystems. Avian haemosporidians have been extensively studied; however, there is limited information on parasites in owls (order Strigiformes). Here, haemosporidians infecting North American owls were characterized using an integrative methodology. Methods: Taking advantage of injured/dead owls from rehabilitation centres in the central and northeastern USA, 53 individuals of Bubo virginianus, Strix varia, Megascops asio, Bubo scandiacus, Aegolius acadicus, and Tyto furcata were screened using polymerase chain reaction for all samples and microscopy for those with available blood smears. Parasite mitochondrial genomes were obtained using a long-read sequencing method (PacBio HiFi), which efficiently detects multiple infections in a single host. The relationships between parasite lineages were estimated using phylogenetic and haplotype network methods. Results: In total, 21 individuals from three species were positive by PCR: B. virginianus (14/17, 82.4%), M. asio (3/5, 60%), and S. varia (4/8, 50%). Two Plasmodium, three Haemoproteus, and four Leucocytozoon lineages were identified infecting these hosts, with one Haemoproteus and one Leucocytozoon being new to science. All positive individuals were infected with Haemoproteus parasites, and two B. virginianus had a mixed infection with Leucocytozoon and Haemoproteus species. The hSTVAR01 cytochrome b (cytb) lineage common in North America is linked for the first time to Haemoproteus syrnii. Haemoproteus syrnii was found in all 14 positive B. virginianus and two S. varia. Notably, all the cytb lineages from previously identified H. syrnii, based on erythrocytic stages, were not monophyletic, indicating the existence of an undescribed species. The pPADOM11 cytb lineage was recognized as an allele of Plasmodium elongatum. Conclusion: Long reads enabled the detection of mixed/co-infections. The link between genetic data and morphospecies was established in two cases. Several Leucocytozoon clades were observed; however, only one morphospecies, Leucocytozoo danilewskyi, has been described in owls. Thus, there is a need for a detailed analysis of blood stages to determine whether different owl Haemoproteus and Leucocytozoon parasites exhibit morphological differences or represent cryptic species. Overall, this study underscores the importance of high-quality molecular data in characterizing the biodiversity of haemosporidian parasites.

Background Avian haemosporidians are single-celled eukaryotic parasites of vertebrates that require dipteran vectors for transmission. The genera Plasmodium , Haemoproteus and Leucocytozoon currently comprise over 5000 parasite lineages based on a 478-bp section of the mitochondrial cytochrome b gene, which is the standard DNA barcode for avian haemosporidians. The mitochondrial genomes of apicomplexan parasites are highly condensed, with a length of approximately 6000 bp, containing three coding genes (cytochrome c oxidase subunit I, cytochrome c oxidase subunit III and cytochrome b ) and dispersed fragments of the small and large ribosomal RNA (rRNA) genes. Since the mitochondrial genomes are relatively conserved, they are valuable markers for studying the phylogenetic relationships between haemosporidian parasites. However, until recently, mitochondrial genomes were unavailable for parasites of the Haemoproteus nisi and Leucocytozoon toddi species groups, which are exclusive to accipitriform raptors and strongly diverged from other Haemoproteus and Leucocytozoon parasites. Methods We screened 171 accipitriform raptors from Austria and Germany using new primers targeting the cytochrome b gene of a previously neglected L. toddi clade. We also developed a new primer assay that enables the amplification and sequencing of the complete mitochondrial genomes of haemosporidian parasites. This process involved long-range PCRs with lineage-specific primers placed within the cytochrome b gene, followed by five nested PCRs targeting conserved sequence regions. Results Screening the accipitriform raptors revealed 10 new L. toddi group lineages. We sequenced 18 mitochondrial genomes belonging to five H. nisi group, nine L. toddi group, and two other Leucocytozoon lineages. Phylogenetic analyses based on mt genome sequences placed the L. toddi lineages within the genus Leucocytozoon , but the results did not support a monophyly of the genus Haemoproteus . Conclusions The new nested PCR approach with lineage-specific primers used for the long-range PCRs described herein successfully enabled the sequencing of the complete mitochondrial genomes, even in samples with mixed infections. The mitochondrial genomes of the H. nisi and L. toddi group lineages are highly valuable for resolving the phylogenetic relationships of the order Haemosporida since these parasites belong to clades distinct from other Haemoproteus and Leucocytozoon parasites. Graphical Abstract

Malaria has been one of the most important diseases of humans throughout history and continues to be a major public health concern. The 5 species of Plasmodium that cause the disease in humans are part of the order Haemosporida, a diverse group of parasites that all have heteroxenous life cycles, alternating between a vertebrate host and a free-flying, blood-feeding dipteran vector. Traditionally, the identification and taxonomy of these parasites relied heavily on life-history characteristics, basic morphological features, and the host species infected. However, molecular approaches to resolving the phylogeny of the group have sometimes challenged many of these traditional hypotheses. One of the greatest debates has concerned the origin of the most virulent of the human-infecting parasites, Plasmodium falciparum, with early results suggesting a close relationship with an avian parasite. Subsequent phylogenetic studies placed it firmly within the mammalian clade instead, but the avian origin hypothesis has been revived with recent genome-based analyses. The rooting of the tree of Haemosporida has also been inconsistent, and the various topologies that result certainly affect our interpretation of the history of the group. There is clearly a pressing need to obtain a much more complete degree of taxon sampling of haemosporidians, as well as a greater number of characters before confidence can be placed in any hypothesis regarding the evolutionary history of the order. There are numerous challenges moving forward, particularly for generating complete genome sequences of avian and saurian parasites.

Leucocytozoon species are cosmopolitan and prevalent avian parasites, with some infections being lethal, mainly due to the exo-erythrocytic development of the parasite in bird tissues. The patterns of exo-erythrocytic development in Leucocytozoon spp. infections in wild birds remain poorly studied. This study investigated the development of Leucocytozoon spp. tissue stages in tits (Paridae). Great tits (Parus major), Blue tits (Cyanistes caeruleus), and Coal tits (Periparus ater) were screened for infections using an integrative approach that consisted of microscopic analysis of thin blood smears, histological techniques, chromogenic in situ hybridization (CISH), PCR-based methods, and phylogenetic analysis. In total, 41 individuals were analyzed (eight naturally infected that were selected and euthanized, and 33 found dead in the wild and opportunistically sampled). Among the naturally infected birds, all individuals that were microscopically positive for Leucocytozoon species were also PCR-positive for these parasites. Co-infections with Plasmodium spp. and Haemoproteus spp. were commonly found, mainly among the opportunistically sampled birds. Two morphotypes were identified, Leucocytozoon majoris (Laveran, 1902) and Leucocytozoon fringillinarum Woodcock, 1910. Tissue stages were present in three birds sampled exclusively during the non-breeding season, two of them with meronts developing in the kidneys and liver, and one individual with a megalomeront in the heart. All the exo-erythrocytic stages were confirmed to be Leucocytozoon spp. by CISH using a Leucocytozoon genus-specific probe. Phylogenetic analysis placed parasite lineages with different morphotypes in separate clades. The developmental patterns of exo-erythrocytic stages of Leucocytozoon spp. in naturally infected passerines are poorly understood, requiring further research.