Comparative analysis of mitochondrial DNA in haemogregarine (Apicomplexa: Adeleorina) blood parasites

Abstract

Southern Africa is recognised as one of the most biodiverse regions globally, with a continual expansion of biodiversity marked by the continuous discovery of new parasites parasitising the country’s rich fauna. Species of Hepatozoon (Adeleorina: Hepatozoidae) comprises a substantial group of parasites, with well over 300 species. Among Southern African frogs, these haemogregarines stand as the most common blood parasites. The considerable diversity within the genus presents a notable phylogenetic challenge due to the limited resolution of the universal 18S rDNA marker in classifying such large groups. The protein-coding genes of the mitochondrial genome have been proposed as markers with higher phylogenetic resolution. In Chapter 2.1 the mitochondrial genomes of three Hepatozoon spp. infecting South African anurans was sequenced through Sanger-sequencing by means of primer walking. The present study yielded three complete mitochondrial genomes, each approximately 6500 base pairs in length, encoding the three protein-coding genes commonly observed in apicomplexan mitochondrial genomes. Supplementary genetic markers, including the ITS-1 region and the 23S pl-rDNA were tested however, the primers used for these markers seem ineffective for the haemogregarines of this study. In conclusion, this study confirms the effectiveness of Sanger sequencing through primer walking for mitochondrial genome sequencing. Furthermore, it underscores the need for improved primers designed specifically for the ITS-1 and 23S markers. Chapter 2.2: The mitochondrial protein-coding genes in species of Haemogregarina (Adeleorina: Haemogregarinidae) have been examined, revealing remarkable variation even among closely related members. Recent research suggests that these protein-coding genes could be valuable for understanding the evolutionary relationships of haemogregarines. However, the unexpectedly high diversity observed among Haemogregarina spp. might have broader implications. In Chapter 2.2, the full mitochondrial genomes of two Haemogregarina spp. were sequenced with Sanger-sequencing by means of primer walking. The two mitochondrial genomes generated resemble that of other Haemogregarina spp. The protein-coding genes' high sequence diversity is consistent with that found in previous studies. Additionally, like other haemogregarine protein-coding genes the COIII gene has the highest sequence diversity compared to the COI and CytB genes. However, it remains unclear whether the sequence diversity of Haemogregarina mitochondrial protein-coding genes reduce their resolution or usefulness in phylogenetic analyses. More mtDNA data will need to be generated for the COI and CytB genes of other Haemogregarina spp. for comprehensive phylogenetic studies to be conducted. Chapter 2.3: Of the Apicomplexans, the genus Hepatozoon (Adeleorina: Haemogregarinidae) has mostly been placed on the parasite back burner in favour of other more medically important parasites. However, Hepatozoon have been recorded in all vertebrate classes and merit more attention. Nevertheless, Hepatozoon spp. are major herpetological parasites, accounting for the majority of reported infections in Southern Africa. Currently, the genus comprises of more than 300 species worldwide of which the phylogenetic placement of certain species have been somewhat of a debate. Although it is evident that the genus needs to be subdivided into multiple genera, the methodology for precisely delineating these divisions remains uncertain. The cytochrome c oxidase subunit I (COI) mitochondrial protein-coding gene sequence has been proposed as an alternative genetic marker to the traditional nuclear 18S rRNA gene sequence. The gene has proven useful in delineating recent speciation events in other apicomplexan parasites. In this study the mitochondrial protein-coding genes COI, cytochrome c oxidase subunit III (COIII), and cytochrome B (CytB) were amplified and sequenced to analyse their ability to resolve recent speciation events within Hepatozoon. A total of 34 protein-coding gene sequences were generated and analysed. The investigation revealed that combining the COI and COIII gene sequences proves most effective in resolving the deeper nodes of the phylogenetic tree. Combining these two gene sequences with the 18S and CytB sequences further aid in resolving these deeper nodes. More data will be needed to determine whether the same conclusions is reached with a larger pool of samples. Considering the challenges posed by the high sequence variability of the COIII gene, it may prove beneficial to undertake whole mitochondrial genome sequencing of Hepatozoon spp., particularly given the difficulties encountered in designing primers for the COIII region.