First Record of Leucocytozoon (Haemosporida: Leucocytozoidae) in Amazonia: Evidence for Rarity in Neotropical Lowlands or Lack of Sampling for This Parasite Genus?

Avian haemosporidians are a widely distributed group of parasites in terms of both avian host associations and geography, infecting nearly all avian orders and inhabiting all continents except for Antarctica (Valkiūnas, 2005). Currently there are more than 250 named species of avian haemosporidians from the genera Haemoproteus, Leucocytozoon, and Plasmodium, all characterized and differentiated morphologically by studying blood films (morphospecies) (see Valkiūnas, 2005). However, the true diversity of this cosmopolitan parasite group is not well established, in part, because of insufficient host sampling across highly diverse regions such as the Neotropics. In the past 2 decades the use of polymerase chain reaction (PCR) for amplification of cytochrome b has revealed many new lineages that are known only from nucleic acid sequence (genetic lineage). Only a few of these genetic lineages have been matched to known morphospecies (Valkiūnas et al., 2008a), with 10-fold more identified genetic lineages (2,828) of avian haemosporidians in the MalAvi database than described morphospecies (Bensch et al., 2009). Efforts to link these genetic lineages to described species are continuing (Valkiūnas et al., 2008a, 2008b), indicating the high diversity of this group as well as the need for further studies using both microscopic examination and molecular analysis.

Leucocytozoon is the least studied haemosporidian genus (see Lutz et al., 2015 and Outlaw et al., 2017 for reviews). However, it is quite diverse in some Neotropical communities and reaches its highest prevalence and diversity in the Peruvian and Colombian highlands above 2,200 and 3,000 meters of altitude, respectively (Galen and Witt, 2014; Lotta et al., 2016), or in high latitudes of the Chilean lowlands (Merino et al., 2008). There are currently more than 50 named species (see Valkiūnas, 2005) and 749 genetic lineages (MalAvi database). The genus Leucocytozoon has 2 subgenera, Leucocytozoon and Akiba, which are distinguished based on the cells in which exo-erythrocytic merogony occurs and by the vectors that transmit them. For the subgenus Leucocytozoon, merogony occurs in hepatocytes and the vectors are blackflies (Diptera: Simuliidae). In contrast, the subgenus Akiba, which includes only Leucocytozoon caulleryi, does not occur in hepatocytes but undergoes merogony in a variety of different host cells and is vectored by biting midges (Diptera: Ceratopogonidae) (Valkiūnas, 2005).

A review by White et al. (1978), published 4 decades ago, summarized the prevalence of avian haemosporidians and other blood parasites in the Neotropics based on records from 35,555 birds (955 species). Remarkably, only 54 individual birds (0.15% prevalence; mostly from Mexico) were infected by Leucocytozoon, followed by Plasmodium (1.9%) and Haemoproteus (7.9%). Before that, Lainson et al. (1970) found no Leucocytozoon in 145 blood slides from 45 bird species from northeast Amazonia. A much larger survey of blood parasites in Brazil a decade later reported only 2 (0.06% prevalence) Leucocytozoon infections in 3,449 individual birds from 195 species sampled from 3 Atlantic Rain Forest sites in São Paulo State (Bennett and Lopes, 1980). The authors of this study concluded, based on their data from Brazil, and with a comparison among 3 regions (Neotropical, Nearctic, and Southeast Asia) that Leucocytozoon was virtually absent in Brazil and rare in the Neotropics (Bennett and Lopes, 1980). A more comprehensive sampling 9 years later in Brazil reported a prevalence of 0.8% for Leucocytozoon (it should be noted that the authors included in their calculation of prevalence Atoxoplasma, Babesia, Hepatozoon, Lankesterella) from 15,574 birds of 266 species collected from altitudes ranging from sea level to 800 meters in the Atlantic Rain Forest (Woodworth-Lynas et al., 1989). Since then, 6 studies and our own unpublished data (Table I) using microscopy failed to find Leucocytozoon in 2,659 birds sampled across 4 Brazilian biomes: Amazonia, the Atlantic Rain Forest, Cerrado, and Pantanal (Table I). Thus, Leucocytozoon has never been found in Brazilian biomes, other than the Atlantic Rain Forest in the 1980s. Although Brazil ranks among countries with the highest diversity of haemosporidian parasites, no DNA sequence data on Leucocytozoon from the Brazilian avifauna have been reported. Currently, the MalAvi database contains 414 lineages of Haemoproteus and Plasmodium recovered from more than 4,000 birds screened in large-scale surveys across the Atlantic Rain Forest and Cerrado (Lacorte et al., 2013) and Amazonia (Fecchio et al., 2017a). Neither of these studies, nor any other published reports using molecular methods (Belo et al., 2011; Fecchio et al., 2013, 2015, 2017b, 2017c; Ferreira Junior et al., 2017), has screened avian samples specifically for the presence of Leucocytozoon.

Table I Summary of studies conducted in Brazil after 1989 using microscopy that failed to find Leucocytozoon and additional surveys using molecular and microscopic screening to detect all 3 haemosporidian genera in Brazilian birds: Haemoproteus, Leucocytozoon, and Plasmodium.

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Blackflies, the primary vectors for Leucocytozoon, can be found in practically all environments, from altitudes ranging from sea level to over 5,000 meters, with the aquatic larvae found in water ranging from very cold (0 C) to rather warm (exceeding 25 C), and varying in salinity (Coscarón and Arias, 2007). The only necessary condition for larval development in this vector family is continuously running water. With this broad range of environments, blackflies are found in a variety of flowing water bodies across the Neotropics, from tiny streams to large rivers (Coscarón and Arias, 2007). This range of environmental conditions is found across all areas where bird communities have been surveyed in Brazil for haemosporidian parasites, as detailed above. In fact, there are more than 550 blackflies species recorded for the New World, 92 of which are known from Brazil (Adler and Crosskey, 2017).

Consequently, 3 questions arise from this short review of Leucocytozoon in Brazilian birds. First, is Leucocytozoon absent in avian communities outside the Atlantic Rain Forest? Second, is the prevalence of this parasite genus so low that even a large-scale survey using molecular screening techniques fails to detect them? Third, why is this parasite genus scarce if its potential vectors are present year-round, in virtually all Brazilian biomes?

To answer the first 2 questions, we molecularly screened 1,261 samples collected from the Brazilian Amazonia and Atlantic Rain Forest biomes for the presence of all 3 haemosporidian genera using either the protocol of Hellgren et al. (2004) or the new method published by Bell et al. (2015) that combines real-time PCR for haemosporidian detection and genus-specific nested PCR of positive samples for identification of haemosporidian taxa. This dataset includes a population of blue-crowned manakins (Lepidothrix coronata) that was previously screened for Plasmodium and Haemoproteus, with 34% of individuals (23 infected out of 67 analyzed) infected by Plasmodium (Bosholn et al., 2016). Although the other samples from Amazonia, the Atlantic Rain Forest, and Pantanal (using microscopic detection for haemosporidians) lacked any Leucocytozoon infections (Table I), we found that 5 of the 90 individuals analyzed from this same population of blue-crowned manakins in central Amazonia were infected with Leucocytozoon. These are the only 5 Leucocytozoon infections known outside of the Atlantic Rain Forest in Brazil. DNA sequencing revealed that all 5 positive samples were infected by a single unique genetic lineage that differs by over 5% from all other known Leucocytozoon morphospecies. Based on this level of genetic divergence this lineage may be a new species (Outlaw and Ricklefs, 2014). The lineage was named LEPCOR08 following standard naming practices (Bensch et al., 2009) and submitted to both GenBank (MG714922–MG714926) and the MalAvi database. Thus, although extremely rare, the genus Leucocytozoon does exist outside of the Atlantic Rain Forest, at least within the Amazonian biome. However, the factors causing the scarcity of Leucocytozoon throughout Brazil are not well understood.

To determine the phylogenetic position of this new lineage within the genus Leucocytozoon a Bayesian inference (BI) phylogeny was reconstructed using Mr. Bayes v.3.2.6 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Available sequences of known morphospecies and representative lineages from the Americas from GenBank and the MalAvi database were aligned using BioEdit v.7.2.5 (Hall, 1999) with Plasmodium elongatum, PADOM09 (KU562554) included as the outgroup. The GTR+I+G model of nucleotide substitution as determined by jModelTest (Guindon and Gascuel, 2003; Darriba et al., 2012) was used for phylogenetic reconstruction with the resulting consensus tree visualized in FigTree v.1.4.3 (Rambaut, 2009).

The new lineage, LEPCOR08, is most closely related to 2 lineages, SETAUD30 and SETAUD31, recovered from the yellow-rumped warbler (Setophaga coronata) in New Mexico, United States (Fig. 1), a species not known to winter in South America (Hunt and Flaspohler, 1998). These 3 lineages form a highly supported clade (BI posterior probability = 1.00) and are nested within a larger clade composed of lineages from both North and South America (Fig. 1), although the monophyly of this larger clade is not strongly supported. The new lineage is not closely related to any of the known morphospecies. The closest morphospecies, Leucocytozoon californicus, recently described by Walther et al. (2016) from American kestrels (Falco sparverius sparverius) in California has 6.4% uncorrected pairwise base pair difference from the new lineage (Fig. 1). The identification of additional Brazilian lineages will be beneficial in understanding the evolutionary history of this parasite genus throughout the Neotropics.

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The third question, about the scarcity of Leucocytozoon, despite its potential vectors being widespread, may be answered by data on parasite development and not solely by data on vector distribution. Plasmodium, Haemoproteus, and Leucocytozoon have different optimal temperature requirements for their development (Valkiūnas, 2005). For example, Roller and Desser (1973) reported a decrease in time needed for exflagellation of Leucocytozoon simondi as the temperature increased from 15 to 26 C. LaPointe et al. (2010) showed that sporogonic development of Plasmodium relictum was completed at temperatures between 17 to 30 C, but development, prevalence, and intensity decreased significantly below 21 C, and complete sporogony was not observed at temperatures below 17 C. Lower temperature not only constrains the sporogonic development of these parasites, but also reduces the activity of potential Leucocytozoon vectors (Valkiūnas, 2005; LaPointe et al., 2010). However, both Neotropical sites where Leucocytozoon was abundant and diverse are associated with low annual average temperature: highlands in Colombia (Lotta et al., 2016) and Peru (Galen and Witt, 2014) and high-latitude lowlands in Chile (Merino et al., 2008). In contrast, the only 2 regions in Brazil where Leucocytozoon has been found, in both cases with very low prevalence and diversity, were the Atlantic Rain Forest (Bennett and Lopes, 1980; Woodworth-Lynas et al., 1989) and Amazonia (this study). These 2 regions, especially Amazonia, have a higher annual average temperature than the highlands of Colombia and Peru and high-latitude lowlands of Chile. Thus, we suggest that higher rather than lower temperature is constraining the distribution of Leucocytozoon across lowland Brazil and perhaps lowland tropical South America. Further spatial analyses using environmental data such as temperature, altitude, latitude, and DNA sequences deposited in the MalAvi database could test whether these variables affect prevalence, diversity, and distribution of Leucocytozoon in birds across South America. However, this will be possible only if researchers studying avian haemosporidians in South America screen for Leucocytozoon as they currently do for Plasmodium and Haemoproteus by using methods that detect all 3 haemosporidian genera in a single assay, such as the restriction-enzyme digestion method described by Beadell and Fleischer (2005) or the real-time PCR method combined with the genus-specific nested-PCR used herein (Bell et al., 2015). Our detection of Leucocytozoon infecting a non-migratory host species in the tropical lowlands suggests that there is a vector capable of transmitting the parasite. This also highlights the importance of conducting studies on vector competence of Brazilian blackflies and sampling the whole avian community to confirm patterns of transmission and host specificity among coexisting vertebrate hosts.