Mitochondrial genome amplification of avian haemosporidian parasites from single-infected wildlife samples using a novel nested PCR approach

Abstract

Haemosporidian parasites that infect birds (Apicomplexa: Haemosporida) are blood parasites that require an invertebrate host (vector) and a vertebrate host for their lifecycle and cause malaria-like diseases. This group of parasites has provided valuable insights into host specificity, virulence, and parasite dispersal. Additionally, they have played a significant role in reshaping our understanding of the evolutionary history of apicomplexans. In order to accurately identify species and to address phylogenetic questions such as the timing of the haemosporidian radiation, the use of a sufficiently large genetic data set is crucial. However, acquiring this genetic data poses significant challenges. In this research, a sensitive nested PCR assay was developed. This assay allows for the easy amplification of complete mitochondrial genomes of haemosporidian parasites in birds, even during the chronic stage of infection. The effectiveness of this new nested PCR assay was evaluated using blood and tissue samples of birds with verified single parasite infections from previous studies. The approach involves amplifying four overlapping fragments of the mitochondrial genome and requires DNA extracts from single-infected samples. This method successfully amplified the complete mitochondrial genomes of 24 distinct haemosporidian parasite lineages found in various bird species. This data is invaluable for conducting phylogenetic analyses and accurately defining species. Furthermore, this study proposes the existence of at least 15 new haemosporidian parasite species based on the genetic information obtained. Data regarding pGRW04, previously categorized as Plasmodium relictum like pSGS1 and pGRW11, indicates that the pGRW04 lineage is actually a separate, hidden Plasmodium species.

Keywords: Haemoproteus; Leucocytozoon; Plasmodium; cox1; cox3; cytb.

Fig. 1

Schematic illustration of the linear mitochondrial genome of Haemoproteus sp. BO6 (KY200985). The three protein coding genes are shaded in black. Genes on the complementary strand are depicted below the line. Overlapping amplification products of the four nested PCRs are given with the associated number

Fig. 2

Agarose gel electrophoresis of nested PCR products of lFOMAD01 from Hypsipetes madagascariensis (Pycnonotidae) from Madagascar. Fragment numbers are given. Lane MR: molecular marker (FastRuler Middle Range DNA Ladder by Thermo Scientific™)

Fig. 3

Phylogenetic relationship of haemosporidian parasites based on concatenated sequences of protein coding regions (cox3, cox1, and cytb). The analysis comprised 53 sequences (3341 bp) of morphospecies (Pacheco et al. 2018b) and newly amplified whole mitochondrial genomes. Values at the nodes are bootstrap values as a percentage obtained for 1000 replicates from a maximum likelihood tree (GTR+G+I). Branch colors indicate the haemosporidian genus. Species names are indicated in bold