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.

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.

Haemosporidia of grey crowned cranes in Rwanda.

Grey crowned cranes (Balearica regulorum) have been facing significant and long-term population declines in East Africa. Studies of Haemosporidian infections are essential to gain insight into pathogenic threats and help infer vector-host relationships, resolve parasite relationships, and support conservation efforts. As part of a program to reintroduce captive cranes in Rwanda back to their natural habitats, through health checks and initial microscopic examination, 120 grey crowned cranes were selected under suspicion of harboring Haemosporidian infections following initial peripheral blood smear examinations. Of these, 104 were infected with Haemoproteus and 3 were coinfected with Leucocytozoon as detected by PCR and microscopy. Sequencing allowed us to identify 2 distinct unreported lineages of Haemoproteus antigonis and one lineage of Leucocytozoon in the subspecies of Grey Crowned Cranes endemic to East Africa, B. r. gibbericeps. Molecularly, our two lineages of Haemoproteus antigonis differ by 32 base pairs and matched with about 95 percent identity to previously reported sequences of H. antigonis found in other species of cranes. No visible morphologic differences were found when compared to images of H. antigonis from previous studies. Our work demonstrates not only a need for increased testing within the family Gruidae, but also to investigate the possibility of cryptic speciation within the morphospecies Haemoproteus antigonis.

Description and molecular characterization of novel Leucocytozoon parasite (Apicomplexa: Haemosporida: Leucocytozoidae), Leucocytozoon polynuclearis n. sp. found in North American woodpeckers.

We describe Leucocytozoon polynuclearis n. sp. (Haemosporida: Leucocytozoidae) from two North American woodpeckers, the northern flicker (Colaptes auratus Linnaeus) and white-headed woodpecker (Dryobates albolarvatus Boie, 1826), based on the morphology of its blood stages and portions of the mitochondrial cytochrome b gene. The most distinctive features of Leucocytozoon polynuclearis n. sp. development are the triangular-shaped host cell nuclei and position of host cell nuclei above gametocytes. This parasite inhabits thrombocytes. Leucocytozoon squamatus Nandi, 1986, the only other Leucocytozoon species detected from Picidae birds, lacks features that are commonly found with L. polynuclearis n. sp. infections. Phylogenetic analysis identified DNA lineages associated with L. polynuclearis n. sp. and showed that this parasite is more closely related to other North American Leucocytozoon species than to L. squamatus, whose initial description was from infected Old World Picidae species. Although there are reports of L. squamatus in North American Picidae species, these detections were based only on microscopic examinations, remain genetically non-characterized, and might be misidentifications with regards to L. polynuclearis n. sp. Available parasite distribution data indicate that L. polynuclearis n. sp. infects Picidae species throughout North America and L. squamatus distribution probably is restricted to Old World Piciformes birds.

A fatal case of a captive snowy owl (Bubo scandiacus) with Haemoproteus infection in Japan.

Parasites of the genus Haemoproteus are vector-borne avian haemosporidia commonly found in bird species of the world. Haemoproteus infections are typically considered relatively benign in birds. However, some Haemoproteus species cause severe disease and mortality, especially for captive birds removed from their original habitat. In September 2018, a captive 15-year-old snowy owl (Bubo scandiacus), kept in a zoological garden of Japan, died subacutely after presenting leg dysfunction. This case showed significantly low PCV and elevated AST, ALT, CK, and LDH values. Many megalomeronts with prominent morphological characteristics of Haemoproteus were observed in the left leg muscles. Those megalomeronts exhibited multilocular structures and were internally filled with merozoites. A new lineage of Haemoproteus was detected by subsequent PCR for the cytochrome b (cytb) gene of avian haemosporidia from DNA extracted from several organ tissues. The detected lineage was classified in the subgenus Parahaemoproteus and was similar to those from the wild birds inhabiting the region including the study area, suggesting that this snowy owl likely acquired its infection from wild birds. This is the first report of a fatal case of a captive bird with a locally transmitted Haemoproteus infection in Japan. We considered the pathogenicity of this infection in conjunction with the clinical course and hematology results. We surmise that snowy owls may be particularly susceptible to infection with Haemoproteus parasites, and warming northern temperatures may exacerbate the overall health of these and other high latitude birds. Further research into the prevalence of Haemoproteus in wild birds near zoological gardens and potential biting midge vectors is necessary for the ex situ conservation of introduced birds.

De novo transcriptome assembly and preliminary analyses of two avian malaria parasites, Plasmodium delichoni and Plasmodium homocircumflexum.

Parasites of the genus Plasmodium infect a wide array of hosts, causing malaria in all major groups of terrestrial vertebrates including primates, reptiles, and birds. Molecular mechanisms explaining why some Plasmodium species are virulent, while other closely related malaria pathogens are relatively benign in the same hosts, remain unclear. Here, we present the RNA sequencing and subsequent transcriptome assembly of two avian Plasmodium parasites which can eventually be used to better understand the genetic mechanisms underlying Plasmodium species pathogenicity in an avian host. Plasmodium homocircumflexum, a cryptic, pathogenic species that often causes mortality and Plasmodium delichoni, a newly described, relatively benign malaria parasite that does not kill its hosts, were used to experimentally infect two Eurasian siskins (Carduelis spinus). RNA extractions were performed and RNA sequencing was completed using high throughput Illumina sequencing. Using established bioinformatics pipelines, the sequencing data from both species were used to generate transcriptomes using published Plasmodium species genomes as a scaffold. The finalized transcriptome of P. homocircumflexum contained 21,612 total contigs while that of P. delichoni contained 12,048 contigs. We were able to identify many genes implicated in erythrocyte invasion actively expressed in both P. homocircumflexum and P. delichoni, including the well described vaccine candidates Apical Membrane Antigen-1 (AMA-1) and Merozoite Surface Protein 1 (MSP1). This work acts as a stepping stone to increase available data on avian Plasmodium parasites, thus enabling future research into the evolution of pathogenicity in malaria.

Delineation of the Genera Haemoproteus and Plasmodium Using RNA-Seq and Multi-gene Phylogenetics.

Members of the order Haemosporida are protist parasites that infect mammals, reptiles and birds. This group includes the causal agents of malaria, Plasmodium parasites, the genera Leucocytozoon and Fallisia, as well as the species rich genus Haemoproteus with its two subgenera Haemoproteus and Parahaemoproteus. Some species of Haemoproteus cause severe disease in avian hosts, and these parasites display high levels of diversity worldwide. This diversity emphasizes the need for accurate evolutionary information. Most molecular studies of wildlife haemosporidians use a bar coding approach by sequencing a fragment of the mitochondrial cytochrome b gene. This method is efficient at differentiating parasite lineages but insufficient for accurate phylogenetic inferences in highly diverse taxa such as haemosporidians. Recent studies have utilized multiple mitochondrial genes (cyt b, cox1 and cox3), sometimes combined with a few apicoplast and nuclear genes. These studies have been highly successful with one notable exception: the evolutionary relationships of the genus Haemoproteus remain unresolved. Here we describe the transcriptome of Haemoproteus columbae and investigate its phylogenetic position recovered from a multi-gene dataset (600 genes). This genomic approach restricts the taxon sampling to 18 species of apicomplexan parasites. We employed Bayesian inference and maximum likelihood methods of phylogenetic analyses and found H. columbae and a representative from the subgenus Parahaemoproteus to be sister taxa. This result strengthens the hypothesis of genus Haemoproteus being monophyletic; however, resolving this question will require sequences of orthologs from, in particular, representatives of Leucocytozoon species.

Avian malaria co-infections confound infectivity and vector competence assays of Plasmodium homopolare.

Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concomitantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare. Song sparrow (Melospiza melodia) blood infected with a low P. homopolare parasitemia was inoculated into a naïve domestic canary (Serinus canaria forma domestica). Within 5 to 10 days post infection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P. homopolare, both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P. homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P. homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P. homopolare DNA. This pattern of mixed Pcat6 and P. homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P. cathemerium lineage (Pcat8); none developed PCR detectable P. homopolare infections. These observations suggest that the original P. homopolare-infected songbird also had two un-detectable P. cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts.

Description and molecular characterization of a new Leucocytozoon parasite (Haemosporida: Leucocytozoidae), Leucocytozoon californicus sp. nov., found in American kestrels (Falco sparverius sparverius).

Diurnal raptors in the order Accipitriformes are commonly parasitized with Leucocytozoon spp., and the prevalence and intensity of parasitemia are often high. However, for raptors in Falconiformes, several studies have reported relatively low prevalences (1 % or less) of Leucocytozoon spp. Leucocytozoon parasite pathogenicity has been documented in falcons, but little is known about the diversity, prevalence, and phylogenetic relationships among Leucocytozoon species in these predatory birds. The research reported here combines molecular and microscopic techniques to identify and describe Leucocytozoon parasites in Falco sparverius sparverius, the American kestrel, and place those parasites into a phylogenetic context with leucocytozoids previously found in other diurnal raptors (Accipitriformes), owls (Strigiformes), passerines (Passeriformes), and other bird species. Of 35 American kestrels sampled, 13 birds (37.1 %) were found by PCR to harbor the DNA lineage of a novel species, Leucocytozoon californicus. No other Leucocytozoon parasite lineages were identified in our sample. Phylogenetic analysis revealed that this parasite clusters more closely with leucocytozoids found in owls and passerines than it does with leucocytozoids found in birds of the genera Buteo and Accipiter of the order Accipitriformes. This is the first described species of Leucocytozoon that parasitizes diurnal raptors in which gametocytes develop exclusively in roundish host blood cells. It is also the first Leucocytozoon species that is described and named in birds of the Falconiformes, in which, for unclear reasons, leucocytozoids are significantly less prevalent and less diverse than in raptors with a similar behavioral ecology belonging to the Accipitriformes.

Genetic sequence data reveals widespread sharing of Leucocytozoon lineages in corvids.

Leucocytozoon, a widespread hemosporidian blood parasite that infects a broad group of avian families, has been studied in corvids (family: Corvidae) for over a century. Current taxonomic classification indicates that Leucocytozoon sakharoffi infects crows and related Corvus spp., while Leucocytozoon berestneffi infects magpies (Pica spp.) and blue jays (Cyanocitta sp.). This intrafamily host specificity was based on the experimental transmissibility of the parasites, as well as slight differences in their morphology and life cycle development. Genetic sequence data from Leucocytozoon spp. infecting corvids is scarce, and until the present study, sequence data has not been analyzed to confirm the current taxonomic distinctions. Here, we predict the phylogenetic relationships of Leucocytozoon cytochrome b lineages recovered from infected American Crows (Corvus brachyrhynchos), yellow-billed magpies (Pica nuttalli), and Steller's jays (Cyanocitta stelleri) to explore the host specificity pattern of L. sakharoffi and L. berestneffi. Phylogenetic reconstruction revealed a single large clade containing nearly every lineage recovered from the three host species, while showing no evidence of the expected distinction between L. sakharoffi and L. berestneffi. In addition, five of the detected lineages were recovered from both crows and magpies. This absence of the previously described host specificity in corvid Leucocytozoon spp. suggests that L. sakharoffi and L. berestneffi be reexamined from a taxonomic perspective.

De novo assembly and transcriptome analysis of Plasmodium gallinaceum identifies the Rh5 interacting protein (ripr), and reveals a lack of EBL and RH gene family diversification.

BACKGROUND: Malaria parasites that infect birds can have narrow or broad host-tropisms. These differences in host specificity make avian malaria a useful model for studying the evolution and transmission of parasite assemblages across geographic ranges. The molecular mechanisms involved in host-specificity and the biology of avian malaria parasites in general are important aspects of malaria pathogenesis that warrant further examination. Here, the transcriptome of the malaria parasite Plasmodium gallinaceum was characterized to investigate the biology and the conservation of genes across various malaria parasite species. METHODS: The P. gallinaceum transcriptome was annotated and KEGG pathway mapping was performed. The ripr gene and orthologous genes that play critical roles in the purine salvage pathway were identified and characterized using bioinformatics and phylogenetic methods. RESULTS: Analysis of the transcriptome sequence database identified essential genes of the purine salvage pathway in P. gallinaceum that shared high sequence similarity to Plasmodium falciparum when compared to other mammalian Plasmodium spp. However, based on the current sequence data, there was a lack of orthologous genes that belonged to the erythrocyte-binding-like (EBL) and reticulocyte-binding-like homologue (RH) family in P. gallinaceum. In addition, an orthologue of the Rh5 interacting protein (ripr) was identified. CONCLUSIONS: These findings suggest that the pathways involved in parasite red blood cell invasion are significantly different in avian Plasmodium parasites, but critical metabolic pathways are conserved throughout divergent Plasmodium taxa.

Coevolutionary patterns and diversification of avian malaria parasites in African sunbirds (Family Nectariniidae).

The coevolutionary relationships between avian malaria parasites and their hosts influence the host specificity, geographical distribution and pathogenicity of these parasites. However, to understand fine scale coevolutionary host-parasite relationships, robust and widespread sampling from closely related hosts is needed. We thus sought to explore the coevolutionary history of avian Plasmodium and the widespread African sunbirds, family Nectariniidae. These birds are distributed throughout Africa and occupy a variety of habitats. Considering the role that habitat plays in influencing host-specificity and the role that host-specificity plays in coevolutionary relationships, African sunbirds provide an exceptional model system to study the processes that govern the distribution and diversity of avian malaria. Here we evaluated the coevolutionary histories using a multi-gene phylogeny for Nectariniidae and avian Plasmodium found in Nectariniidae. We then assessed the host-parasite biogeography and the structuring of parasite assemblages. We recovered Plasmodium lineages concurrently in East, West, South and Island regions of Africa. However, several Plasmodium lineages were recovered exclusively within one respective region, despite being found in widely distributed hosts. In addition, we inferred the biogeographic history of these parasites and provide evidence supporting a model of biotic diversification in avian Plasmodium of African sunbirds.

Transcriptome sequencing and analysis of Plasmodium gallinaceum reveals polymorphisms and selection on the apical membrane antigen-1.

BACKGROUND: Plasmodium erythrocyte invasion genes play a key role in malaria parasite transmission, host-specificity and immuno-evasion. However, the evolution of the genes responsible remains understudied. Investigating these genes in avian malaria parasites, where diversity is particularly high, offers new insights into the processes that confer malaria pathogenesis. These parasites can pose a significant threat to birds and since birds play crucial ecological roles they serve as important models for disease dynamics. Comprehensive knowledge of the genetic factors involved in avian malaria parasite invasion is lacking and has been hampered by difficulties in obtaining nuclear data from avian malaria parasites. Thus the first Illumina-based de novo transcriptome sequencing and analysis of the chicken parasite Plasmodium gallinaceum was performed to assess the evolution of essential Plasmodium genes. METHODS: White leghorn chickens were inoculated intravenously with erythrocytes containing P. gallinaceum. cDNA libraries were prepared from RNA extracts collected from infected chick blood and sequencing was run on the HiSeq2000 platform. Orthologues identified by transcriptome sequencing were characterized using phylogenetic, ab initio protein modelling and comparative and population-based methods. RESULTS: Analysis of the transcriptome identified several orthologues required for intra-erythrocytic survival and erythrocyte invasion, including the rhoptry neck protein 2 (RON2) and the apical membrane antigen-1 (AMA-1). Ama-1 of avian malaria parasites exhibits high levels of genetic diversity and evolves under positive diversifying selection, ostensibly due to protective host immune responses. CONCLUSION: Erythrocyte invasion by Plasmodium parasites require AMA-1 and RON2 interactions. AMA-1 and RON2 of P. gallinaceum are evolutionarily and structurally conserved, suggesting that these proteins may play essential roles for avian malaria parasites to invade host erythrocytes. In addition, host-driven selection presumably results in the high levels of genetic variation found in ama-1 of avian Plasmodium species. These findings have implications for investigating avian malaria epidemiology and population dynamics. Moreover, this work highlights the P. gallinaceum transcriptome as an important public resource for investigating the diversity and evolution of essential Plasmodium genes.

Description, molecular characterization, and patterns of distribution of a widespread New World avian malaria parasite (Haemosporida: Plasmodiidae), Plasmodium (Novyella) homopolare sp. nov.

Plasmodium (Novyella) homopolare, a newly described Plasmodium species, was found in a wide range of Passeriformes species in California, USA, and Colombia. This parasite infected more than 20% of the sampled bird community (N = 399) in California and was found in 3.6% of birds sampled (N = 493) in Colombia. Thus far, it has been confirmed in North and South America where it is present in numerous species of migratory and resident birds from six families. Based on 100% matches, or near-100% matches (i.e., ≤2-nucleotide difference), to DNA sequences previously deposited in GenBank, this parasite is likely also distributed in the Eastern USA, Central America, and the Caribbean. Here, we describe the blood stages of P. homopolare and its mtDNA cytochrome b sequence. P. homopolare belongs to the subgenus Novyella and can be readily distinguished from the majority of other Novyella species, primarily, by the strictly polar or subpolar position of meronts and advanced trophozoites in infected erythrocytes. We explore possible reasons why this widespread parasite has not been described in earlier studies. Natural malarial parasitemias are usually light and co-infections predominate, making the parasites difficult to detect and identify to species when relying exclusively on microscopic examination of blood films. The combined application of sequence data and digital microscopy techniques, such as those used in this study, provides identifying markers that will facilitate the diagnosis of this parasite in natural avian populations. We also address the evolutionary relationship of this parasite to other species of Plasmodium using phylogenetic reconstruction.

The Wildlife Malaria Research network (WIMANET): Meeting report on the 1st WIMANET workshop.

The Wildlife Malaria Network (WIMANET) is a groundbreaking multinational collaboration focused on studying vector-borne haemosporidian parasites in wildlife. Unlike human malaria, wildlife malaria is found on all continents except Antarctica, with parasites being transmitted by a range of vectors. The complexity and diversity of these parasites makes it necessary to have an interdisciplinary approach to understand and mitigate their impacts. Established in 2023 within the framework of COST-Action (European Cooperation in Science and Technology), WIMANET unites researchers from diverse scientific backgrounds to tackle critical questions about wildlife malaria on a global scale. This meeting report summarises the activities and plans resulting from the 1st meeting of WIMANET's six working groups, spanning the genetic and morphological identification of parasites to understanding the drivers of host-parasite-vector associations from individual to community levels. WIMANET's collaborative efforts aim to fill the knowledge gaps and foster large-scale research initiatives transcending local and regional boundaries.

Identification and expression of maebl, an erythrocyte-binding gene, in Plasmodium gallinaceum.

Avian malaria is of significant ecological importance and serves as a model system to study broad patterns of host switching and host specificity. The erythrocyte invasion mechanism of the malaria parasite Plasmodium is mediated, in large part, by proteins of the erythrocyte-binding-like (ebl) family of genes. However, little is known about how these genes are conserved across different species of Plasmodium, especially those that infect birds. Using bioinformatical methods in conjunction with polymerase chain reaction (PCR) and genetic sequencing, we identified and annotated one member of the ebl family, merozoite apical erythrocyte-binding ligand (maebl), from the chicken parasite Plasmodium gallinaceum. We then detected the expression of maebl in P. gallinaceum by PCR analysis of cDNA isolated from the blood of infected chickens. We found that maebl is a conserved orthologous gene in avian, mammalian, and rodent Plasmodium species. The duplicate extracellular binding domains of MAEBL, responsible for erythrocyte binding, are the most conserved regions. Our combined data corroborate the conservation of maebl throughout the Plasmodium genus and may help elucidate the mechanisms of erythrocyte invasion in P. gallinaceum and the host specificity of Plasmodium parasites.

Molecular characterization of Haemoproteus sacharovi (Haemosporida, Haemoproteidae), a common parasite of columbiform birds, with remarks on classification of haemoproteids of doves and pigeons.

Haemoproteus (Haemosporida, Haemoproteidae) is the largest genus of avian haemosporidian parasites, some species of which cause lethal diseases in birds. Subgenera Parahaemoproteus and Haemoproteus are usually accepted in this genus; these parasites are transmitted by biting midges (Ceratopogonidae) and hippoboscid flies (Hippoboscidae), respectively. As of yet, species of Parahaemoproteus have not been reported to infect doves and pigeons (Columbiformes), parasites of these birds have not been reported to be transmitted by biting midges (Ceratopogonidae). Applying microscopy and PCR based methods, we identified mitochondrial cytochrome b (cyt b) sequences of Haemoproteus sacharovi, a wide-spread parasite of doves and pigeons. Phylogenetic relationships of dove haemoproteids, which traditionally have been classified in the subgenus Haemoproteus, showed that H. sacharovi and H. turtur, common parasites of doves, branch in the clade with Parahaemoproteus species, indicating that these haemoproteids may belong to this subgenus and are likely transmitted by biting midges. This study provides barcodes for H. sacharovi, clarifies the taxonomic positions of H. sacharovi and H. turtur, and indicates directions for development of classification of avian haemoproteid species. Our analysis shows that the current subgeneric classification of avian haemoproteids is generally effective, but the position of some species may need to be revised.

Host and habitat specialization of avian malaria in Africa.

Studies of both vertebrates and invertebrates have suggested that specialists, as compared to generalists, are likely to suffer more serious declines in response to environmental change. Less is known about the effects of environmental conditions on specialist versus generalist parasites. Here, we study the evolutionary strategies of malaria parasites (Plasmodium spp.) among different bird host communities. We determined the parasite diversity and prevalence of avian malaria in three bird communities in the lowland forests in Cameroon, highland forests in East Africa and fynbos in South Africa. We calculated the host specificity index of parasites to examine the range of hosts parasitized as a function of the habitat and investigated the phylogenetic relationships of parasites. First, using phylogenetic and ancestral reconstruction analyses, we found an evolutionary tendency for generalist malaria parasites to become specialists. The transition rate at which generalists become specialists was nearly four times as great as the rate at which specialists become generalists. We also found more specialist parasites and greater parasite diversity in African lowland rainforests as compared to the more climatically variable habitats of the fynbos and the highland forests. Thus, with environmental changes, we anticipate a change in the distribution of both specialist and generalist parasites with potential impacts on bird communities.

Novel RNA viruses associated with avian haemosporidian parasites.

Avian haemosporidian parasites can cause malaria-like symptoms in their hosts and have been implicated in the demise of some bird species. The newly described Matryoshka RNA viruses (MaRNAV1 and MaRNAV2) infect haemosporidian parasites that in turn infect their vertebrate hosts. MaRNAV2 was the first RNA virus discovered associated with parasites of the genus Leucocytozoon. By analyzing metatranscriptomes from the NCBI SRA database with local sequence alignment tools, we detected two novel RNA viruses; we describe them as MaRNAV3 associated with Leucocytozoon and MaRNAV4 associated with Parahaemoproteus. MaRNAV3 had ~59% amino acid identity to the RNA-dependent RNA-polymerase (RdRp) of MaRNAV1 and ~63% amino acid identity to MaRNAV2. MaRNAV4 had ~44% amino acid identity to MaRNAV1 and ~47% amino acid identity to MaRNAV2. These findings lead us to hypothesize that MaRNAV_like viruses are widespread and tightly associated with the order Haemosporida since they have been described in human Plasmodium vivax, and now two genera of avian haemosporidians.

Evaluation of minimally invasive sampling methods for detecting Avipoxvirus: Hummingbirds as a case example.

Avian pox is a common avian virus that in its cutaneous form can cause characteristic lesions on a bird's dermal surfaces. Detection of avian pox in free-ranging birds historically relied on observations of visual lesions and/or histopathology, both which can underestimate avian pox prevalence. We compared traditional visual observation methods for avian pox with molecular methods that utilize minimally invasive samples (blood, toenail clipping, feathers, and dermal swabs) in an ecologically important group of birds, hummingbirds. Specifically, avian pox prevalence in several species of hummingbirds were examined across multiple locations using three different methods: (1) visual inspection of hummingbirds for pox-like lesions from a long-term banding data set, (2) qPCR assay of samples from hummingbird carcasses from wildlife rehabilitation centers, and (3) qPCR assay of samples from live-caught hummingbirds. A stark difference in prevalences among these three methods was identified, with an avian pox prevalence of 1.5% from banding data, 20.4% from hummingbird carcasses, and 32.5% from live-caught hummingbirds in California. This difference in detection rates underlines the necessity of a molecular method to survey for avian pox, and this study establishes one such method that could be applied to other wild bird species. Across all three methods, Anna's hummingbirds harbored significantly higher avian pox prevalence than other species examined, as did males compared with females and birds caught in Southern California compared with Northern California. After hatch-year hummingbirds also harbored higher avian pox prevalences than hatch-year hummingbirds in the California banding data set and the carcass data set. This is the first study to estimate the prevalence of avian pox in hummingbirds and address the ecology of this hummingbird-specific strain of avian pox virus, providing vital information to inform future studies on this charismatic and ecologically important group of birds.