Co-infection with Leucocytozoon and Other Haemosporidian Parasites Increases with Latitude and Altitude in New World Bird Communities.

Establishing how environmental gradients and host ecology drive spatial variation in infection rates and diversity of pathogenic organisms is one of the central goals in disease ecology. Here, we identified the predictors of concomitant infection and lineage richness of blood parasites in New Word bird communities. Our multi-level Bayesian models revealed that higher latitudes and elevations played a determinant role in increasing the probability of a bird being co-infected with Leucocytozoon and other haemosporidian parasites. The heterogeneity in both single and co-infection rates was similarly driven by host attributes and temperature, with higher probabilities of infection in heavier migratory host species and at cooler localities. Latitude, elevation, host body mass, migratory behavior, and climate were also predictors of Leucocytozoon lineage richness across the New World avian communities, with decreasing parasite richness at higher elevations, rainy and warmer localities, and in heavier and resident host species. Increased parasite richness was found farther from the equator, confirming a reverse Latitudinal Diversity Gradient pattern for this parasite group. The increased rates of Leucocytozoon co-infection and lineage richness with increased latitude are in opposition with the pervasive assumption that pathogen infection rates and diversity are higher in tropical host communities.

Distinct biogeographic processes and areas of endemism contributed differentially to Plasmodium and Parahaemoproteus community assembly on Marajó Island.

Amazonia is the primary source of haemosporidian diversity for South American biomes. Yet, our understanding of the contribution of each area of endemism and the biogeographical processes that generated such diversity in this group of vector transmitted parasites remains incomplete. For example, a recently formed fluvial island in the Amazon delta - Marajó Island, is composed of avian lineages from adjacent Amazonian areas of endemism, but also from open habitats, such as Cerrado. This raises the question: Is the parasite assemblage found in avian hosts on this island formed by parasite lineages from adjacent Amazonian areas of endemism or Cerrado? Here, we assessed the spatiotemporal evolution of Plasmodium and Parahaemoproteus parasites. Our biogeographic analysis showed that dispersal dominated Plasmodium diversification, whereas duplication was more frequent for the genus Parahaemoproteus. We show that the Inambari area of endemism was the primary source for Plasmodium diversity on Marajó Island, but that this island received more Parahaemoproteus lineages from Cerrado than any Amazonian area of endemism. The unique patterns of dispersal for each parasite genus coupled with their propensity to shift hosts locally may have facilitated their diversification across Amazonia, suggesting that differences in deep evolutionary history may have constrained their colonization of Marajó Island.

Host foraging behavior and nest type influence prevalence of avian haemosporidian parasites in the Pantanal.

Avian haemosporidians from the genera Plasmodium and Haemoproteus are vector transmitted parasites. A growing body of evidence suggests that variation in their prevalence within avian communities is correlated with a variety of avian ecological traits. Here, we examine the relationship between infection probability and diversity of haemosporidian lineages and avian host ecological traits (average body mass, foraging stratum, migratory behavior, and nest type). We used molecular methods to detect haemosporidian parasites in blood samples from 642 individual birds of 149 species surveyed at four localities in the Brazilian Pantanal. Based on cytochrome b sequences, we recovered 28 lineages of Plasmodium and 17 of Haemoproteus from 31 infected avian species. Variation in lineage diversity among bird species was not explained by avian ecological traits. Prevalence was heterogenous across avian hosts. Bird species that forage near the ground were less likely to be infected by Haemoproteus, whereas birds that build open cup nests were more likely infected by Haemoproteus. Furthermore, birds foraging in multiple strata were more likely to be infected by Plasmodium. Two other ecological traits, often related to host resistance (body mass and migratory behavior), did not predict infection probability among birds sampled in the Pantanal. Our results suggest that avian host traits are less important determinants of haemosporidian diversity in Pantanal than in other regions, but reinforces that host attributes, related to vector exposure, are to some extent important in modulating infection probability within an avian host assemblage.

Loss of forest cover and host functional diversity increases prevalence of avian malaria parasites in the Atlantic Forest.

Host phylogenetic relatedness and ecological similarity are thought to contribute to parasite community assembly and infection rates. However, recent landscape level anthropogenic changes may disrupt host-parasite systems by impacting functional and phylogenetic diversity of host communities. We examined whether changes in host functional and phylogenetic diversity, forest cover, and minimum temperature influence the prevalence, diversity, and distributions of avian haemosporidian parasites (genera Haemoproteus and Plasmodium) across 18 avian communities in the Atlantic Forest. To explore spatial patterns in avian haemosporidian prevalence and taxonomic and phylogenetic diversity, we surveyed 2241 individuals belonging to 233 avian species across a deforestation gradient. Mean prevalence and parasite diversity varied considerably across avian communities and parasites responded differently to host attributes and anthropogenic changes. Avian malaria prevalence (termed herein as an infection caused by Plasmodium parasites) was higher in deforested sites, and both Plasmodium prevalence and taxonomic diversity were negatively related to host functional diversity. Increased diversity of avian hosts increased local taxonomic diversity of Plasmodium lineages but decreased phylogenetic diversity of this parasite genus. Temperature and host phylogenetic diversity did not influence prevalence and diversity of haemosporidian parasites. Variation in the diversity of avian host traits that promote parasite encounter and vector exposure (host functional diversity) partially explained the variation in avian malaria prevalence and diversity. Recent anthropogenic landscape transformation (reduced proportion of native forest cover) had a major influence on avian malaria occurrence across the Atlantic Forest. This suggests that, for Plasmodium, host phylogenetic diversity was not a biotic filter to parasite transmission as prevalence was largely explained by host ecological attributes and recent anthropogenic factors. Our results demonstrate that, similar to human malaria and other vector-transmitted pathogens, prevalence of avian malaria parasites will likely increase with deforestation.

An inverse latitudinal gradient in infection probability and phylogenetic diversity for Leucocytozoon blood parasites in New World birds.

Geographic variation in environmental conditions as well as host traits that promote parasite transmission may impact infection rates and community assembly of vector-transmitted parasites. Identifying the ecological, environmental and historical determinants of parasite distributions and diversity is therefore necessary to understand disease outbreaks under changing environments. Here, we identified the predictors and contributions of infection probability and phylogenetic diversity of Leucocytozoon (an avian blood parasite) at site and species levels across the New World. To explore spatial patterns in infection probability and lineage diversity for Leucocytozoon parasites, we surveyed 69 bird communities from Alaska to Patagonia. Using phylogenetic Bayesian hierarchical models and high-resolution satellite remote-sensing data, we determined the relative influence of climate, landscape, geography and host phylogeny on regional parasite community assembly. Infection rates and parasite diversity exhibited considerable variation across regions in the Americas. In opposition to the latitudinal gradient hypothesis, both the diversity and prevalence of Leucocytozoon parasites decreased towards the equator. Host relatedness and traits known to promote vector exposure neither predicted infection probability nor parasite diversity. Instead, the probability of a bird being infected with Leucocytozoon increased with increasing vegetation cover (NDVI) and moisture levels (NDWI), whereas the diversity of parasite lineages decreased with increasing NDVI. Infection rates and parasite diversity also tended to be higher in cooler regions and higher latitudes. Whereas temperature partially constrains Leucocytozoon diversity and infection rates, landscape features, such as vegetation cover and water body availability, play a significant role in modulating the probability of a bird being infected. This suggests that, for Leucocytozoon, the barriers to host shifting and parasite host range expansion are jointly determined by environmental filtering and landscape, but not by host phylogeny. Our results show that integrating host traits, host ancestry, bioclimatic data and microhabitat characteristics that are important for vector reproduction are imperative to understand and predict infection prevalence and diversity of vector-transmitted parasites. Unlike other vector-transmitted diseases, our results show that Leucocytozoon diversity and prevalence will likely decrease with warming temperatures.

Bird Tissues from Museum Collections Are Reliable for Assessing Avian Haemosporidian Diversity.

Birds harbor a diverse group of haemosporidian parasites that reproduce and develop in the host blood cells, muscle tissue, and various organs, which can cause negative effects on the survival and reproduction of their avian hosts. Characterization of the diversity, distribution, host specificity, prevalence patterns, and phylogenetic relationships of these parasites is critical to the study of avian host-parasite ecology and evolution and for understanding and preventing epidemics in wild bird populations. Here, we tested whether muscle and liver samples collected as part of standard ornithological museum expeditions can be examined to study the diversity and distributions of haemosporidians in the same way as blood collected from individual birds that are typically banded and released. We used a standard molecular diagnostic screening method for mitochondrial DNA (cytochrome b) of the parasites and found that blood, muscle, and liver collected from the same host individual provide similar estimates of prevalence and diversity of haemosporidians from the genera Parahaemoproteus and Leucocytozoon. Although we found higher prevalence for the genus Plasmodium when we screened blood vs. liver and muscle samples, the estimates of the diversity of Plasmodium from different tissue types are not affected at the community level. Given these results, we conclude that for several reasons existing museum genetic resources collections are valuable data sources for the study of haemosporidians. First, ornithological museum collections around the world house tens of thousands of vouchered tissue samples collected from remote regions of the world. Second, the host specimens are vouchered and thus host identification and phenotype are permanently documented in databased archives with a diversity of associated ancillary data. Thus, not only can identifications be confirmed but also a diversity of morphological measurements and data can be measured and accessed for these host specimens in perpetuity.

Avian host composition, local speciation and dispersal drive the regional assembly of avian malaria parasites in South American birds.

Identifying the ecological factors that shape parasite distributions remains a central goal in disease ecology. These factors include dispersal capability, environmental filters and geographic distance. Using 520 haemosporidian parasite genetic lineages recovered from 7,534 birds sampled across tropical and temperate South America, we tested (a) the latitudinal diversity gradient hypothesis and (b) the distance-decay relationship (decreasing proportion of shared species between communities with increasing geographic distance) for this host-parasite system. We then inferred the biogeographic processes influencing the diversity and distributions of this cosmopolitan group of parasites across South America. We found support for a latitudinal gradient in diversity for avian haemosporidian parasites, potentially mediated through higher avian host diversity towards the equator. Parasite similarity was correlated with climate similarity, geographic distance and host composition. Local diversification in Amazonian lineages followed by dispersal was the most frequent biogeographic events reconstructed for haemosporidian parasites. Combining macroecological patterns and biogeographic processes, our study reveals that haemosporidian parasites are capable of circumventing geographic barriers and dispersing across biomes, although constrained by environmental filtering. The contemporary diversity and distributions of haemosporidian parasites are mainly driven by historical (speciation) and ecological (dispersal) processes, whereas the parasite community assembly is largely governed by host composition and to a lesser extent by environmental conditions.

A soluble peripherin/Rds C-terminal polypeptide promotes membrane fusion and changes conformation upon membrane association.

Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content.

Association of a photoreceptor-specific tetraspanin protein, ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes.

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.