Evolution of Plastic Transmission Strategies in Avian Malaria

Malaria parasites have been shown to adjust their life history traits to changing environmental conditions. Parasite relapses and recrudescences—marked increases in blood parasite numbers following a period when the parasite was either absent or present at very low levels in the blood, respectively—are expected to be part of such adaptive plastic strategies. Here, we first present a theoretical model that analyses the evolution of transmission strategies in fluctuating seasonal environments and we show that relapses may be adaptive if they are concomitant with the presence of mosquitoes in the vicinity of the host. We then experimentally test the hypothesis that Plasmodium parasites can respond to the presence of vectors. For this purpose, we repeatedly exposed birds infected by the avian malaria parasite Plasmodium relictum to the bites of uninfected females of its natural vector, the mosquito Culex pipiens, at three different stages of the infection: acute (∼34 days post infection), early chronic (∼122 dpi) and late chronic (∼291 dpi). We show that: (i) mosquito-exposed birds have significantly higher blood parasitaemia than control unexposed birds during the chronic stages of the infection and that (ii) this translates into significantly higher infection prevalence in the mosquito. Our results demonstrate the ability of Plasmodium relictum to maximize their transmission by adopting plastic life history strategies in response to the availability of insect vectors.     

Malaria infection increases bird attractiveness to uninfected mosquitoes

The epidemiology of vector‐borne pathogens is largely determined by the host‐choice behaviour of their vectors. Here, we investigate whether a Plasmodium infection renders the host more attractive to host‐seeking mosquitoes. For this purpose, we work on a novel experimental system: the avian malaria parasite Plasmodium relictum, and its natural vector, the mosquito Culex pipiens. We provide uninfected mosquitoes with a choice between an uninfected bird and a bird undergoing either an acute or a chronic Plasmodium infection. Mosquito choice is assessed by microsatellite typing of the ingested blood. We show that chronically infected birds attract significantly more vectors than either uninfected or acutely infected birds. Our results suggest that malaria parasites manipulate the behaviour of uninfected vectors to increase their transmission. We discuss the underlying mechanisms driving this behavioural manipulation, as well as the broader implications of these effects for the epidemiology of malaria.     

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.     

Parasitological and new molecular-phylogenetic characterization of the malaria parasite Plasmodium tejerai in South American penguins

This study is the first report on mortality of Spheniscus magellanicus, penguin of South America, caused by Plasmodium tejerai, which was identified using morphological and molecular analyses. Blood stages (trophozoites, meronts and gametocytes) were reported and illustrated. The necropsy revealed marked splenomegaly and pulmonary edema, as well as moderate hepatomegaly and hydropericardium. The histopathology revealed the presence of tissue meronts in the macrophages and endothelial cells of multiple organs. The molecular analyses showed 5.6% of genetic divergence in cytochrome b gene between P. tejerai and Plasmodium relictum. Morphology of blood and tissue stages of P. tejerai is similar to P. relictum; the distinction between these two species requires experience in the identification of avian Plasmodium species. Molecular studies associated with reliably identified morphological species are useful for barcoding and comparisons with previous studies of wildlife malaria infections as well as for posterior phylogenetic and phylogeographic studies. S. magellanicus is a new host record of P. tejerai, which is the virulent parasite and worth more attention in avian conservation and veterinary medicine projects in South America.     

The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite

Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2(-) sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.     

Mhc supertypes confer both qualitative and quantitative resistance to avian malaria infections in a wild bird population

Major histocompatibility complex (Mhc) genes are believed to play a key role in the genetic basis of disease control. Although numerous studies have sought links between Mhc and disease prevalence, many have ignored the ecological and epidemiological aspects of the host–parasite interaction. Consequently, interpreting associations between prevalence and Mhc has been difficult, whereas discriminating alleles for qualitative resistance, quantitative resistance and susceptibility remains challenging. Moreover, most studies to date have quantified associations between genotypes and disease status, overlooking the complex relationship between genotype and the properties of the Mhc molecule that interacts with parasites. Here, we address these problems and demonstrate avian malaria (Plasmodium) parasite species-specific associations with functional properties of Mhc molecules (Mhc supertypes) in a wild great tit (Parus major) population. We further show that correctly interpreting these associations depends crucially on understanding the spatial variation in risk of infection and the fitness effects of infection. We report that a single Mhc supertype confers qualitative resistance to Plasmodium relictum, whereas a different Mhc supertype confers quantitative resistance to Plasmodium circumflexum infections. Furthermore, we demonstrate common functional properties of Plasmodium-resistance alleles in passerine birds, suggesting this is a model system for parasite–Mhc associations in the wild.     

Local prevalence and transmission of avian malaria in the Alakai Plateau of Kauai,Hawaii, U.S.A.

Avian malaria is among the most important threats to native Hawaiian forest birds. It is caused by the parasite Plasmodium relictum and is transmitted by the introduced mosquito vector Culex quinquefasciatus. Temperature increases and precipitation declines due to climate change over the last decade may be responsible for the observed recent expansion in the range and prevalence of avian malaria on the Alakai Plateau, Kauai Island. To examine the hypothesis that conditions are now favorable for transmission of malaria on the Plateau, mosquitoes were sampled with CO₂ and Reiter oviposition traps at three sites (Kawaikoi, Halepa'akai, and Koke'e) on several occasions between October, 2013 and April, 2014. P. relictum infection was assessed by PCR or dissection under a microscope. We also surveyed mosquito larvae along Halepa'akai and Kawaikoi streams. We observed that Cx. quinquefasciatus is well established on the Alakai Plateau, as mosquitoes were caught on all field trips, except in April at Halepa'akai, and larvae were found throughout the year. We observed differences in adult abundance among sites and microhabitats (stream vs ridge lines).     

A rare case of Plasmodium (Haemamoeba) relictum infection in a free-living Red Knot (Calidris canutus rufa, Scolopacidae)

The Red Knot (Calidris canutus rufa) is a Nearctic migrant shorebird that breeds in the Canadian Arctic and spends the winter season in coastal sites in South America. A rare case of a blood protozoan was found by molecular analyses from an adult bird captured during spring migration at the last refuelling stopover in Delaware Bay USA in 2006. The parasite was identified as Plasmodium relictum belonging to subgenus Haemamoeba based on the shape of meronts, roundish gametocytes, and its position in the erythrocytes from the blood smears examination. A partial cytochrome b sequence was a 100% match to a sequence of Plasmodium relictum, sequence Genbank accession number: id DQ659543.1 (lineage code haplotype P5). This is the first report of avian malaria in a wild individual of C. c. rufa.     

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

A comprehensive study reveals that kinesins in the malaria parasite Plasmodium have diverse cellular roles and locations, including functions in spindle assembly during proliferation, axoneme formation in flagellum biogenesis, and determining the apical morphology of the cell.     

A highly invasive malaria parasite has expanded its range to non-migratory birds in North America

Parasite range expansions are a direct consequence of globalization and are an increasing threat to biodiversity. Here, we report a recent range expansion of the SGS1 strain of a highly invasive parasite, Plasmodium relictum, to two non-migratory passerines in North America. Plasmodium relictum is considered one of the world''s most invasive parasites and causes the disease avian malaria: this is the first reported case of SGS1 in wild non-migratory birds on the continent. Using a long-term database where researchers report avian malaria parasite infections, we summarized our current understanding of the geographical range of SGS1 and its known hosts. We also identified the most likely geographical region of this introduction event using the MSP1 allele. We hypothesize that this introduction resulted from movements of captive birds and subsequent spillover to native bird populations, via the presence of competent vectors and ecological fitting. Further work should be conducted to determine the extent to which SGS1 has spread following its introduction in North America.     

Different paths – the same virulence: experimental study on avian single and co-infections with Plasmodium relictum and Plasmodium elongatum

Co-infections are prevalent worldwide, however, we are still struggling to understand interactions between different parasites and their impacts on host fitness. In the present experimental study we analysed the infection dynamics of two avian malarial parasites Plasmodium elongatum (genetic lineage pERIRUB01) and Plasmodium relictum (genetic lineage pSGS1) and their impacts on host health during single and co-infections. We reveal that P. elongatum intensity of parasitemia is enhanced by the presence of P. relictum during co-infection, while the parasitemia of P. relictum stays the same. This illustrates how development of a parasite (P. elongatum) which infects both mature and young (polychromatic) red blood cells (RBCs) is facilitated during co-infection with a parasite which specialises in adult RBCs only (P. relictum). The virulence of co-infections was similar to that of the more virulent parasite (P. elongatum). However, the profile of infection and the mechanisms that caused mortality were different. Birds infected with P. elongatum only start to die due to non-regenerative anaemia, when intensity of parasitemia is light and the number of polychromatic RBCs decrease dramatically. Meanwhile, co-infected birds start to die when the mean intensity of parasitemia reaches 10% and the number of polychromatic RBCs increases abnormally, reflecting regenerative anaemia. Our findings reveal that typically measured parameters of virulence (e.g., mortality rate, level of hematocrit) can be the same during single and co-infections, but the mechanisms responsible for the observed virulence can be different. This information serves a better understanding of the processes underpinning the interactions of co-infected parasite species.     

Anopheles aquasalis Infected by Plasmodium vivax Displays Unique Gene Expression Profiles when Compared to Other Malaria Vectors and Plasmodia

Malaria affects 300 million people worldwide every year and is endemic in 22 countries in the Americas where transmission occurs mainly in the Amazon Region. Most malaria cases in the Americas are caused by Plasmodium vivax, a parasite that is almost impossible to cultivate in vitro, and Anopheles aquasalis is an important malaria vector. Understanding the interactions between this vector and its parasite will provide important information for development of disease control strategies. To this end, we performed mRNA subtraction experiments using A. aquasalis 2 and 24 hours after feeding on blood and blood from malaria patients infected with P. vivax to identify changes in the mosquito vector gene induction that could be important during the initial steps of infection. A total of 2,138 clones of differentially expressed genes were sequenced and 496 high quality unique sequences were obtained. Annotation revealed 36% of sequences unrelated to genes in any database, suggesting that they were specific to A. aquasalis. A high number of sequences (59%) with no matches in any databases were found 24 h after infection. Genes related to embryogenesis were down-regulated in insects infected by P. vivax. Only a handful of genes related to immune responses were detected in our subtraction experiment. This apparent weak immune response of A. aquasalis to P. vivax infection could be related to the susceptibility of this vector to this important human malaria parasite. Analysis of some genes by real time PCR corroborated and expanded the subtraction results. Taken together, these data provide important new information about this poorly studied American malaria vector by revealing differences between the responses of A. aquasalis to P. vivax infection, in relation to better studied mosquito-Plasmodium pairs. These differences may be important for the development of malaria transmission-blocking strategies in the Americas.     

Molecular Genetics Evidence for the in Vivo Roles of the Two Major NADPH-dependent Disulfide Reductases in the Malaria Parasite

Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in an oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. Here, we dissected the in vivo roles of both redox pathways by gene targeting of the respective NADPH-dependent disulfide reductases. We show that Plasmodium berghei glutathione reductase and thioredoxin reductase are dispensable for proliferation of the pathogenic blood stages. Intriguingly, glutathione reductase is vital for extracellular parasite development inside the insect vector, whereas thioredoxin reductase is dispensable during the entire parasite life cycle. Our findings suggest that glutathione reductase is the central player of the parasite redox network, whereas thioredoxin reductase fulfils a specialized and dispensable role for P. berghei. These results also indicate redundant roles of the Plasmodium redox pathways during the pathogenic blood phase and query their suitability as promising drug targets for antimalarial intervention strategies.     

Parallel evolution of gene classes,but not genes: Evidence from Hawai'ian honeycreeper populations exposed to avian malaria

Adaptation in nature is ubiquitous, yet characterizing its genomic basis is difficult because population demographics cause correlations with nonadaptive loci. Introduction events provide opportunities to observe adaptation over known spatial and temporal scales, facilitating the identification of genes involved in adaptation. The pathogen causing avian malaria, Plasmodium relictum, was introduced to Hawai'i in the 1930s and elicited extinctions and precipitous population declines in native honeycreepers. After a sharp initial population decline, the Hawai'i ‘amakihi (Chlorodrepanis virens) has evolved tolerance to the parasite at low elevations where P. relictum exists, and can sustain infection without major fitness consequences. High‐elevation, unexposed populations of ‘amakihi display little to no tolerance. To explore the genomic basis of adaptation to P. relictum in low‐elevation ‘amakihi, we genotyped 125 ‘amakihi from the island of Hawai'i via hybridization capture to 40,000 oligonucleotide baits containing SNPs and used the reference ‘amakihi genome to identify genes potentially under selection from malaria. We tested for outlier loci between low‐ and high‐elevation population pairs and identified loci with signatures of selection within low‐elevation populations. In some cases, genes commonly involved in the immune response (e.g., major histocompatibility complex) were associated with malaria presence in the population. We also detected several novel candidate loci that may be implicated in surviving malaria infection (e.g., beta‐defensin, glycoproteins and interleukin‐related genes). Our results suggest that rapid adaptation to pathogens may occur through changes in different immune genes, but in the same classes of genes, across populations.