High lineage diversity and host sharing of malarial parasites in a local avian assemblage

Bird populations often have high prevalences of the haemosporidians Haemoproteus spp. and Plasmodium spp., but the extent of host sharing and host switching among these parasite lineages and their avian hosts is not well known. While sampling within a small geographic region in which host individuals are likely to have been exposed to the same potential parasite lineages, we surveyed highly variable mitochondrial DNA from haemosporidians isolated from 14 host taxa representing 4 avian families (Hirundinidae, Parulidae, Emberizidae, and Fringillidae). Analyses of cytochrome b sequences from 83 independent infections identified 29 unique haplotypes, representing 2 well-differentiated Haemoproteus spp. lineages and 6 differentiated Plasmodium spp. lineages. A phylogenetic reconstruction of relationships among these lineages provided evidence against host specificity at the species and family levels, as all haemosporidian lineages recovered from 2 or more host individuals (2 Haemoproteus and 3 Plasmodium lineages) were found in at least 2 host families. We detected a similar high level of host sharing; the 3 most intensively sampled host species each harbored 4 highly differentiated haemosporidian lineages. These results indicate that some Haemoproteus spp. and Plasmodium spp. lineages exhibit a low degree of host specificity, a phenomenon with implications for ecological and evolutionary interactions among these parasites and their hosts.     

Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds

A fragment of the mitochondrial cytochrome b gene of avian malaria (genera Haemoproteus and Plasmodium) was amplified from blood samples of 12 species of passerine birds from the genera Acrocephalus, Phylloscopus and Parus. By sequencing 478 nucleotides of the obtained fragments, we found 17 different mitochondrial haplotypes of Haemoproteus or Plasmodium among the 12 bird species investigated. Only one out of the 17 haplotypes was found in more than one host species, this exception being a haplotype detected in both blue tits (Parus caeruleus) and great tits (Parus major). The phylogenetic tree which was constructed grouped the sequences into two clades, most probably representing Haemoproteus and Plasmodium, respectively. We found two to four different parasite mitochondrial DNA (mtDNA) haplotypes in four bird species. The phylogenetic tree obtained from the mtDNA of the parasites matched the phylogenetic tree of the bird hosts poorly. For example, the two tit species and the willow warbler (Phylloscopus trochilus) carried parasites differing by only 0.6% sequence divergence, suggesting that Haemoproteus shift both between species within the same genus and also between species in different families. Hence, host shifts seem to have occurred repeatedly in this parasite host system. We discuss this in terms of the possible evolutionary consequences for these bird species.     

Variation in host specificity between species of avian hemosporidian parasites: evidence from parasite morphology and cytochrome B gene sequences

A parasite's shift to a new host may have serious evolutionary consequences, since host switching usually is associated with a change in virulence and may lead to the evolution of emerging diseases. This phenomenon remains insufficiently studied in wildlife. Here, we combine microscopic examination of blood films and PCR-based methods to investigate the natural host specificity of Haemoproteus and Plasmodium spp. in birds of 4 families of the Passeriformes within a small geographic area. The material was collected on the Curonian Spit in the Baltic Sea between May and July in 2003-2004. A nested-PCR protocol was used for amplifying and sequencing a fragment of 480 nucleotides of the cytochrome b gene of the mtDNA of these parasites. Blood samples from 282 birds, which were positive both by microscopic examination of blood films and mtDNA amplification, were used in this study. We found that Haemoproteus majoris (lineages hPARUS1, hCCF5, hWW2, and hPHSIB1), Haemoproteus sp. (hWW1), Plasmodium (Haemamoeba) sp. (pSGS1), and Plasmodium (Haemamoeba) sp. (pGRW11) are capable of infecting birds belonging to different families of passeriform birds. Some species of Haemoproteus are less specific than have been traditionally believed. Haemoproteus majoris appears to have a genetic predisposition to have a broad host range. The level of host specificity varies markedly among different species of hemosporidian parasites of birds. The natural host range is thus not a reliable taxonomic character in the systematics of these parasites in the form in which it is still accepted in some recent taxonomic studies.     

Prevalence of avian haemosporidian parasites and their host fidelity in the central Philippine islands

We examined the prevalence and host fidelity of avian haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon and Plasmodium in the central Philippine islands by sampling 23 bird families (42 species). Using species-specific PCR assays of the mitochondrial cytochrome b gene (471base pairs, bp), we detected infections in 91 of the 215 screened individuals (42%). We also discriminated between single and multiple infections. Thirty-one infected individuals harbored a single Haemoproteus lineage (14%), 18 a single Leucocytozoon lineage (8%) and 12 a single Plasmodium lineage (6%). Of the 215 screened birds, 30 (14%) presented different types of multiple infections. Intrageneric mixed infections were generally more common (18 Haemoproteus/Haemoproteus, 3 Leucocytozoon/Leucocytozoon, and 1 Plasmodium/Plasmodium) than intergeneric mixed infections (7 Haemoproteus/Leucocytozoon and 1 Haemoproteus/Leucocytozoon/Plasmodium). We recovered 81 unique haemosporidian mitochondrial haplotypes. These clustered in three strongly supported monophyletic clades that correspond to the three haemosporidian genera. Related lineages of Haemoproteus and Leucocytozoon were more likely to derive from the same host family than predicted by chance; however, this was not the case for Plasmodium. These results indicate that switches between host families are more likely to occur in Plasmodium. We conclude that Haemoproteus has undergone a recent diversification across well-supported host-family specific clades, while Leucocytozoon shows a longer association with its host(s). This study supports previous evidence of a higher prevalence and stronger host-family specificity of Haemoproteus and Leucocytozoon compared to Plasmodium.     

A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences

A phylogeny of haemosporidian parasites (phylum Apicomplexa, family Plasmodiidae) was recovered using mitochondrial cytochrome b gene sequences from 52 species in 4 genera (Plasmodium, Hepatocystis, Haemoproteus, and Leucocytozoon), including parasite species infecting mammals, birds, and reptiles from over a wide geographic range. Leucocytozoon species emerged as an appropriate out-group for the other malarial parasites. Both parsimony and maximum-likelihood analyses produced similar phylogenetic trees. Life-history traits and parasite morphology, traditionally used as taxonomic characters, are largely phylogenetically uninformative. The Plasmodium and Hepatocystis species of mammalian hosts form 1 well-supported clade, and the Plasmodium and Haemoproteus species of birds and lizards form a second. Within this second clade, the relationships between taxa are more complex. Although jackknife support is weak, the Plasmodium of birds may form 1 clade and the Haemoproteus of birds another clade, but the parasites of lizards fall into several clusters, suggesting a more ancient and complex evolutionary history. The parasites currently placed within the genus Haemoproteus may not be monophyletic. Plasmodium falciparum of humans was not derived from an avian malarial ancestor and, except for its close sister species, P. reichenowi, is only distantly related to haemospordian parasites of all other mammals. Plasmodium is paraphyletic with respect to 2 other genera of malarial parasites, Haemoproteus and Hepatocystis. Explicit hypothesis testing supported these conclusions.     

Haemosporidian vector research: marriage of molecular and microscopical approaches is essential

Many species of malaria and related haemosporidian parasites (Haemosporida) are responsible for diseases in wild and domestic animals. These pathogens are exclusively transmitted by blood-sucking dipteran insects (Diptera). Traditional vector studies, which are based mainly on experimental infection and subsequent dissection of insects, are time-consuming, so progress in the identification of the vectors has been slow. Since the discovery of haemosporidians in wildlife by V. Danilewsky in 1884, it took over 70 years to determine the main vector groups of these parasites. However, precise vector-parasite relationships remain insufficiently investigated in wildlife, particularly at the species level of haemosporidians and their vectors. Molecular tools have provided innovative opportunities to speed such research. In this issue of Molecular Ecology, Martínez-de la Puente et al. (2011) collected, for the first time, a significant PCR-based set of data on the presence of lineages of the pigment-forming haemosporidians (species of Haemoproteus and Plasmodium) in biting midges (Culicoides). They identified numerous associations between Culicoides spp. and Haemoproteus spp., indicating directions for future targeting vector studies of haemoproteids.     

Seasonal variation in abundance and diversity of eavesdropping frog‐biting midges (Diptera,Corethrellidae) in a neotropical rainforest

1. In the tropics, precipitation patterns result in seasonal fluctuations in the abundance and distribution of plant and animal species. Tropical predators and parasites are therefore faced with seasonal changes in prey and host availability. 2. This study investigates the seasonal interaction among a specialised ectoparasite, eavesdropping frog‐biting midges (Corethrella spp.), and their anuran hosts, examining how the abundance and diversity of the frog‐biting midge community fluctuate between the rainy (host abundant) and dry (host sparse) seasons. 3. Midges were captured in both the rainy and dry seasons using acoustic playbacks of calls from a common frog species that breeds during the rainy season, the túngara frog (Engystomops, Physalaemus, pustulosus). During the dry season túngara frog choruses are absent. To explore seasonal shifts in host preference or changes in the midge community due to host specificity, midges were also captured using playbacks of calls from a frog that breeds during the dry season, the pug‐nosed tree frog (Smilisca sila). 4. While the overall abundance of midges decreased in the dry season, only slight differences in the relative abundance between midge species were found. These results suggest that midge populations can shift between hosts as they become available across seasons, allowing adult populations of frog‐biting midges to persist year‐round. To overcome the challenge of detecting and localising different host species, it is proposed that frog‐biting midges have evolved a generalised acoustic template, allowing them to respond to a broad range of available hosts, regardless of seasonal host composition.     

Prevalence and diversity of avian hematozoan parasites in Asia: a regional survey

Tissue samples from 699 birds from three regions of Asia (Myanmar, India, and South Korea) were screened for evidence of infection by avian parasites in the genera Plasmodium and Haemoproteus. Samples were collected from November 1994 to October 2004. We identified 241 infected birds (34.0%). Base-on-sequence data for the cytochrome b gene from 221 positive samples, 34 distinct lineages of Plasmodium, and 41 of Haemoproteus were detected. Parasite diversity was highest in Myanmar followed by India and South Korea. Parasite prevalence differed among regions but not among host families. There were four lineages of Plasmodium and one of Haemoproteus shared between Myanmar and India and only one lineage of Plasmodium shared between Myanmar and South Korea. No lineages were shared between India and South Korea, although an equal number of distinct lineages were recovered from each region. Migratory birds in South Korea and India originate from two different migratory flyways; therefore cross-transmission of parasite lineages may be less likely. India and Myanmar shared more host species and habitat types compared to South Korea. Comparison between low-elevation habitat in India and Myanmar showed a difference in prevalence of haematozoans.     

Bloodmeal analysis reveals avian Plasmodium infections and broad host preferences of Culicoides (Diptera: Ceratopogonidae) vectors

Changing environmental conditions and human encroachment on natural habitats bring human populations closer to novel sources of parasites, which might then develop into new emerging diseases. Diseases transmitted by host generalist vectors are of special interest due to their capacity to move pathogens into novel hosts. We hypothesize that humans using forests for recreation are exposed to a broad range of parasites from wild animals and their vectors. A corollary of this is that new vector-host, parasite-host, and vector-parasite associations could eventually develop. Thus, we expect to observe atypical vector-host associations. Using molecular bloodmeal analysis via amplification of the mtDNA COI gene we identified the vertebrate hosts of Culicoides (Diptera: Ceratopogonidae) species in a sub-urban forest of Southwestern Germany. Bloodmeals were also checked for haemosporidian infections by amplifying a fragment of the mtDNA cyt b gene. We identified a total of 20 Culicoides species, thirteen of which fed on humans. From 105 screened bloodmeals we obtained high quality sequences for 77 samples, 73 (94.8%) originated from humans, two from livestock (Bos taurus and Equus caballus), and two from wild birds (Sylvia atricapilla and Turdus merula). We found that four Culicoides species previously assumed to feed exclusively on either birds (C. kibunensis) or domestic mammals (C. chiopterus, C. deltus, C. scoticus) fed also on humans. A total of six Culicoides abdomens were infected with avian haemosporidian parasites (Plasmodium or Haemoproteus), four of those abdomens contained blood derived from humans. Our results suggest that parasites of wild animals may be transferred to humans through infectious bites of Culicoides vectors. Further, we show that Culicoides vectors believed to be a specialist on specific vertebrate groups can have plastic feeding preferences, and that Culicoides are susceptible to infection by Plasmodium parasites, though vector viability must still be experimentally demonstrated.     

Widespread and structured distributions of blood parasite haplotypes across a migratory divide of the Swainson's thrush (Catharus ustulatus)

We examined the phylogenetic distribution of cytochrome b haplotypes of the avian blood parasite genera Haemoproteus and Plasmodium across the migratory divide of the Swainson's thrush (Catharus ustulatus) in British Columbia, Canada. From 87 host individuals, we identified 8 parasite haplotypes; 4 of Plasmodium and 4 of Haemoproteus. Six haplotypes were novel; 1 Haemoproteus haplotype was identical to H. majoris found in the blue tit (Parus caeruleus) in Sweden, and another halotype was identical to a Plasmodium haplotype found in the white-crowned sparrow (Zonotrichia leucophrys) in Oregon. The 2 most abundant parasite haplotypes were widely distributed across the contact zone, whereas 2 other parasite haplotypes seem to have structured distributions. Compared with 74 Plasmodium and Haemoproteus haplotypes published in GenBank, haplotypes recovered from Swainson's thrushes do not form monophyletic groups, and they are closely related to haplotypes from a variety of other hosts and localities. In addition, we recovered 2 Swainson's thrush Plasmodium haplotypes from the nonmigratory orange-billed nightingale thrush (Catharus aurantiirostris) in Costa Rica. This study is the first to elucidate avian blood parasite transmission, distribution, and phylogenetic relationships in an avian contact zone in North America.     

Molecular characterization of malarial parasites in captive passerine birds

Seven of 28 passerine birds that died in captivity were positive for malarial parasites by polymerase chain reaction targeting the mitochondrial cytochrome b (cytB) and apicoplast ribosomal RNA (rRNA) genes. Each bird was infected with a single parasite lineage having a unique genotype. Apicoplast rRNA sequences were present both in Haemoproteus spp. and Plasmodium spp. and had typically high adenosine + thymidine content. Phylogenies for cytB and apicoplast rRNA sequences were largely congruent and supported previous studies that suggest that Plasmodium-Haemoproteus spp. underwent synchronous speciation with their avian hosts, interrupted by sporadic episodes of host switching. Apicoplast phylogeny further indicated that Haemoproteus spp. are ancestral to Plasmodium spp. All the 7 infected passerine birds had histologic lesions of malaria, and malarial parasites may have contributed to the death of at least 4 animals. These findings provide new genetic data on passerine hematozoa, including initial sequences of apicoplast DNA, and emphasize the relevance of parasite prevalence, evolutionary relationships, and host switching to modern management and husbandry practices of captive birds.     

Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector-borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites ( Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon ) from these sites in two widespread species of African rainforest birds, the yellow-whiskered greenbul ( Andropadus latirostris , Pycnonotidae) and the olive sunbird ( Cyanomitra olivacea , Nectariniidae). Twenty-six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats ( Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea , as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host–parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.     

Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest

Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector-host-parasite system is essential. Little is known about avian parasite-vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR-based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild-caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex and Mansonia) were infected with twenty-two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one-third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate-host breadth. This study suggests extensive invertebrate host shifts in mosquito-parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species.     

Haemosporidian blood parasites in European birds of prey and owls

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.     

Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region

The degree to which widespread avian blood parasites in the genera Plasmodium and Haemoproteus pose a threat to novel hosts depends in part on the degree to which they are constrained to a particular host or host family. We examined the host distribution and host-specificity of these parasites in birds from two relatively understudied and isolated locations: Australia and Papua New Guinea. Using polymerase chain reaction (PCR), we detected infection in 69 of 105 species, representing 44% of individuals surveyed (n = 428). Across host families, prevalence of Haemoproteus ranged from 13% (Acanthizidae) to 56% (Petroicidae) while prevalence of Plasmodium ranged from 3% (Petroicidae) to 47% (Ptilonorhynchidae). We recovered 78 unique mitochondrial lineages from 155 sequences. Related lineages of Haemoproteus were more likely to derive from the same host family than predicted by chance at shallow (average LogDet genetic distance = 0, n = 12, P = 0.001) and greater depths (average distance = 0.014, n = 11, P < 0.001) within the parasite phylogeny. Within two major Haemoproteus subclades identified in a maximum likelihood phylogeny, host-specificity was evident up to parasite genetic distances of 0.029 and 0.007 based on logistic regression. We found no significant host relationship among lineages of Plasmodium by any method of analysis. These results support previous evidence of strong host-family specificity in Haemoproteus and suggest that lineages of Plasmodium are more likely to form evolutionarily-stable associations with novel hosts.     

Presence of Plasmodium and Haemoproteus in breeding prothonotary warblers (Protonotaria citrea: Parulidae): temporal and spatial trends in infection prevalence

Prothonotary warbler (Protonotaria citrea) has shown a long-term decline in abundance in the United States. As a long-range migrant, these warblers are exposed to parasites in both tropical and temperate regions. The focus of this study was to use molecular techniques to examine the temporal prevalence patterns of heamosopridian parasites Plasmodium and Haemoproteus in breeding prothonotary warblers. The prevalence (presence or absence) of Plasmodium and Haemoproteus species was assayed using primer sets for the cytochrome b gene of the mitochondrial DNA. Blood samples were obtained from 187 adult prothonotary warblers collected at 3 central Virginia, U.S.A., breeding sites. The relationship between haemosporidian parasite infections and reproductive success also was examined. We found that 71% of captured prothonotary warblers were infected with haemosporidian parasites, specifically, with 36% prevalence for Haemoproteus spp. and 44% prevalence for Plasmodium spp., during the 2008 breeding season; for both parasites, prevalence increased throughout the season. We found significant variation in haemosporidian parasite prevalence across the breeding season that was strongly site specific. Conversely, we found no significant effects of haemosporidian parasite infections on the reproductive success of prothonotary warblers. This is in sharp contrast to recent reports suggesting considerable effects of these parasites on the reproductive success of wild birds.     

Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.     

The Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) Is Capable of Developing Late Stage Infections of Leishmania enriettii

BackgroundDespite their importance in animal and human health, the epidemiology of species of the Leishmania enriettii complex remains poorly understood, including the identity of their biological vectors. Biting midges of the genus Forcipomyia (Lasiohelea) have been implicated in the transmission of a member of the L. enriettii complex in Australia, but the far larger and more widespread genus Culicoides has not been investigated for the potential to include vectors to date.Conclusions/SignificanceWe demonstrate that L. enriettii can develop late stage infections in the biting midge Culicoides sonorensis. This midge was found to be susceptible to L. enriettii to a similar degree as Lutzomyia longipalpis, the vector of Leishmania infantum in South America. Our results support the hypothesis that some biting midges could be natural vectors of the L. enriettii complex because of their vector competence, although not Culicoides sonorensis itself, which is not sympatric, and midges should be assessed in the field while searching for vectors of related Leishmania species including L. martiniquensis and "L. siamensis".