Primers targeting mitochondrial genes of avian haemosporidians: PCR detection and differential DNA amplification of parasites belonging to different genera

Abstract

Haemosporida is a diverse group of vector-borne parasitic protozoa, ubiquitous in terrestrial vertebrates worldwide. The renewed interest in their diversity has been driven by the extensive use of molecular methods targeting mitochondrial genes. Unfortunately, most studies target a 478 bp fragment of the cytochrome b (cytb) gene, which often cannot be used to separate lineages from different genera found in mixed infections that are common in wildlife. In this investigation, an alignment constructed with 114 mitochondrial genome sequences belonging to four genera (Leucocytozoon, Haemoproteus, Plasmodium and Hepatocystis) was used to design two different sets of primers targeting the cytb gene as well as the other two mitochondrial DNA genes: cytochrome c oxidase subunit 1 and cytochrome c oxidase subunit 3. The design of each pair of primers required consideration of different criteria, including a set for detection and another for differential amplification of DNA from parasites belonging to different avian haemosporidians. All pairs of primers were tested in three laboratories to assess their sensitivity and specificity under diverse practices and across isolates from different genera including single and natural mixed infections as well as experimental mixed infections. Overall, these primers exhibited high sensitivity regardless of the differences in laboratory practices, parasite species, and parasitemias. Furthermore, those primers designed to separate parasite genera showed high specificity, as confirmed by sequencing. In the case of cytb, a nested multiplex (single tube PCR) test was designed and successfully tested to differentially detect lineages of Plasmodium and Haemoproteus parasites by yielding amplicons with different sizes detectable in a standard agarose gel. To our knowledge, the designed assay is the first test for detection and differentiation of species belonging to these two genera in a single PCR. The experiments across laboratories provided recommendations that can be of use to those researchers seeking to standardise these or other primers to the specific needs of their field investigations.

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

“…To summarize previous research, four articles supported a Parahaemoproteus /subgenus Haemoproteus sister relationship (Valkiūnas et al 2010, 2016; Palinauskas et al 2015; Lutz et al 2016), five articles supported a Plasmodium / Parahaemoproteus sister relationship (Martinsen et al 2008; Santiago-Alarcón et al 2010; Martinezde la Puente et al 2011; Pacheco et al 2018) and one article obtained mixed results depending on the taxa, evolutionary model and genes analyzed (Borner et al 2016). Pacheco et al (2018) bears special mention as the most recent study with the largest dataset in terms of complete mitochondrial genes (Pacheco et al 2018). With relatively few deep-sequenced haemosporidian taxa available, we can only speculate as to the difference such a selection can have on phylogenetic inference.…”

“…Recent work by Valkiūnas describing a new malaria parasite Plasmodium delichoni included phylogenetic analyses describing a sister relationship of the subgenus Haemoproteus and Parahaemoproteus , and a more distant relationship with Plasmodium (Valkiūnas et al 2016). Finally, work by Pacheco et al (2018) used 114 complete mitochondrial genomes of many taxa to infer the relationships of haemosporidians for the genera Leucocytozoon, Plasmodium, Haemoproteus and Hepatocystis . These phylogenetic reconstructions based on both Bayesian and likelihood methods found a sister relationship between Plasmodium and Parahaemoproteus .…”

“…Additionally, many studies also focused only on cyt b analyses, specifically a 478 bp sequence of cyt b . Pacheco et al (2018) stress the necessity of expanding data collection standards beyond sequencing only the cyt b gene (Pacheco et al 2018). Our attempt to clarify the relationships between the subgenus Haemoproteus and Parahaemoproteus by using a large number of nuclear genes strengthens the claim of a monophyletic Haemoproteus clade.…”

“…The primary limitation of such methods is that genomic and transcriptomic datasets, required for phylogenomic analyses, are particularly difficult to obtain from haemosporidian parasites (Lauron et al 2014; Bensch et al 2016; Videvall et al 2017; Böehme et al 2018; Videvall 2018). Recent multi-gene phylogenetic analyses of the avian apicomplexans performed with multiple statistical methods produced well-resolved phylogenies with one exception in the case of haemosporidian parasites, i.e., the placement of subgenus Haemoproteus in relation to subgenus Parahaemoproteus and genus Plasmodium (Martinsen et al 2008; Bensch et al 2016; Borner et al 2016; Pacheco et al 2018). An early multi-gene study by Martinsen et al (2008) showed the genus Haemoproteus as paraphyletic, with Plasmodium forming a sister relationship with subgenus Parahaemoproteus .…”

“…Furthermore, the relationships of the Haemoproteus subgenera have been open to debate, with one subgenus forming a sister relationship with Plasmodium as in Fig. 1a (Martinsen et al 2008; Santiago-Alarcón et al 2010; Martinez-de la Puente et al 2011; Pacheco et al 2018) or with both subgenera forming a monophyletic clade as in Fig. 1b (Valkiūnas et al 2010, 2016; Levin et al 2012; Carlson et al 2013; Palinauskas et al 2015; Lutz et al 2016).…”