In the present study, we have successfully developed a novel nested PCR assay designed to amplify entire mitochondrial genomes of avian haemosporidian parasites. This methodology was evaluated using distinct sets of samples possessing varying levels of DNA quality from wild birds. Encouragingly, complete mitochondrial genomes were effectively amplified from both frozen tissue samples and small blood samples preserved in lysis buffer.
The established nested PCR approach facilitates the amplification of complete mitochondrial genomes from avian hosts captured in the wild, even during the chronic infection phase. Notably, this approach presents a simplified alternative to other PCR methodologies that rely on cloning, resource-intensive whole genomic DNA amplification, or intricate sample preparation (e.g. Pacheco et al. 2018; Böhme et al. 2018). An essential prerequisite for successful amplification is the use of a single infected sample. For identifying single infections, we recommend the multiplex PCR developed by Ciloglu et al. (2023), enabling the simultaneous detection of avian haemosporidian parasites across all three genera, including the subgenera Haemoproteus and Parahaemoproteus. Alternatively, the widely employed nested PCR assay by Hellgren et al. (2004) for specific amplification of Plasmodium/Haemoproteus and Leucocytozoon cytb barcodes, combined with the multiplex PCR by Pacheco et al. (2018a) for detecting mixed infections with Plasmodium and Haemoproteus, can be utilized. The multiplex PCR by Ciloglu et al. (2023) offers the advantage of streamlined execution and furnishes a robust indicator of multiple infections. Conversely, the combination of the methods by Hellgren et al (2004) and Pacheco (2018a) yields not only identification of single infections but also a wealth of additional data, notably the parasite barcodes.
Through the new PCR approach, it emerged that many samples previously identified as single infections in prior studies (Musa et al. 2022; Magaña Vázquez et al. 2022; Schmid et al 2017) in fact contained mixed infections. Notably, the tissue samples from Carrion Crows displayed a higher prevalence of multiple Leucocytozoon lineage infections (> 65%) than initially anticipated (Schmid et al. 2017). Within these samples, successful amplification of the complete mitochondrial genome was only feasible for one lineage (lCOCOR09), as the majority (19 out of 20) demonstrated mixed infections. The capacity to identify parasites at the lineage level is restricted to fragment 3, covering the cytb barcode. When other fragments deviate from fragment 3 in overlapping regions, detection of mixed infections is possible, but precise identification of the additional parasite lineage is impeded due to data comparability limitations. Enhanced data availability in the future may enable identification of individual fragments and the resolution of mixed infections within genera.
Due to a lower amount of mixed infections, mitochondrial genome amplification was notably more successful using DNA extracts from tissue samples of Eurasian Magpies and blood samples from Malagasy birds stored in lysis buffer. Remarkably, over 55% of the lineages yielded successful amplification of their entire mitochondrial genomes. This achievement has yielded a substantial corpus of new genetic data, significantly enriching the dataset for future phylogenetic inquiries.
The reconstructed haemosporidian phylogeny closely mirrors the structure of the most recent hypothesis (Pacheco and Escalante 2023). Leucocytozoon species underpin the phylogenetic tree, with the exception of Leucocytozoon (Akiba) caulleryi, which forms a sister-clade to the subgenus Parahaemoproteus. This affinity may stem from shared vector usage, notably biting midges (Ceratopogonidae). Within the Leucocytozoon cluster, the newly discovered lineages from Madagascar and Germany demonstrate no close ties to previously described morphospecies, suggesting the potential presence of undescribed or genetically unlinked morphospecies. Further investigations should address this intriguing observation.
In the Haemoproteus (Parahaemoproteus) subtree, lineage hFOUMAD02 potentially represents an uncharacterized species. A distinct clade encompassing lineages hNEWBR04, hNEWBR05, and hNEWAM04 presents intriguing similarity, with hNEWAM04 differing by 31–33 base pairs, while hNEWBR04 and hNEWBR05 vary by just four base pairs out of a total of 3,317. Morphological variations observed by Magaña Vázquez et al. (2022) in the gametocytes of these lineages suggest the likelihood of novel species. The postulation that hNEWAM04 might correspond to the already defined Haemoproteus vangii (Savage et al. 2009) necessitates formal description in future studies.
Most Plasmodium lineages exhibit limited association with previously documented morphospecies, indicating potential novel species. pCOPALB03 appears closely linked to P. homopolare, a widespread New World parasite of Passeriformes (Walther et al. 2014). However, the sequences differ in 46 of 3,317 base pairs, and distinctions in the meront morphology (Magaña Vázquez et al. 2022) suggest pCOPALB03 might constitute a separate species.
Plasmodium relictum stands out due to its ubiquitous distribution and extensive range of avian hosts and mosquito vectors. Within this species, five lineages (pSGS1, pGRW4, pGRW11, pLZFUS01, and pPHCOL01) were identified and partially characterized. These closely related lineages are indistinct morphologically and often cannot be differentiated using vector or blood stage morphology (Valkiunas et al. 2018). Unlike most other Plasmodium parasites, transmission of P. relictum pSGS1 takes place as far north as northern Norway (Marzal et al. 2011). In Europe, the findings of the lineage pGRW04 are restricted to tropical migratory birds after they return from winter quarters, suggesting the absence of active transmission on breeding grounds (Martínez-de la Puente et al. 2021). The reported differences in geographical distribution of the lineages pSGS1 and pGRW11 on the one hand, and GRW04 on the other hand are difficult to explain bearing in mind the enormously broad range of their susceptible avian hosts and globally distributed mosquito vector species, such as Culex pipiens and Culex quinquefasciatus. Looking at partial sequences of merozoite surface protein 1 (msp1) gene revealed differences in five alleles were revealed between the lineage pGRW04 and the lineages pSGS1 and pGRW11, suggesting the lack of gene flow between those parasites (Hellgren et al. 2015). Furthermore, preliminary observations indicate that several European bird species can resist pGRW04 strains, which were isolated from African migrating Great read warblers Acrocephalus arundinaceus (Dimitrov et al. 2015). This data indicates that the lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 might belong to the same P. relictum morphotype, but some of them also might represent cryptic species of the P. relictum group. Phylogenetic analysis of whole mitochondrial genomes previously contained only sequences of pSGS1 (KY653773) and pGRW11 (KY653772). This study's phylogenetic tree now includes data from pGRW04 and the quite similar pFOUMAD03 (21 bp difference of a total of 5997 bp). The branch containing sequences of pSGS1 and pGRW11 appears as sister-clade to the branch of pGRW04 and pFOUMAD03. Based on the concatenated sequence of protein coding genes (3,317 bp), the lineages differ in 43–52 base pairs. Because of this clear separation of pGRW04 and from pSGS1 in terms of genetic data and transmission areas, it is strongly suggested that these lineages be considered cryptic species.
In summary, our newly introduced PCR protocol enables the amplification of complete mitochondrial genomes from avian haemosporidian parasites. The assay provides a streamlined approach to obtaining extensive genetic data even from single infected wild bird samples with mild parasitemia. This dataset proves pivotal for future phylogenetic analyses and species delimitation, as exemplified by our findings for pGRW04.