Heteroplasmy in Nematode parasites of veterinary importance: Spirocerca lupi.

Milanzie, M.*, Joubert, F., Pierneef, R., De Waal, P.

Spirocerca lupi is a parasitic nematode that causes spirocercosis in both wild and domestic dogs. An understanding of the parasite’s evolutionary relationships, epidemiology, and ecology is important for developing effective strategies to control its spread and infection rate. Heteroplasmy, the presence of multiple mitochondrial DNA (mtDNA) variants within a cell or an individual, can interfere with population genetics and phylogenetic studies that heavily rely on mtDNA markers. Furthermore, any erroneous analysis conducted on mtDNA due to heteroplasmy could cause researchers to interpret the data as evidence of the emergence of a new cryptic species. Therefore, this study estimated the extent of heteroplasmy in the different genes of the Spirocerca lupi mitochondrial genome. This study represents a continuation of the previous study conducted by Rothmann-Meyer et al. (2021). Paired-end Illumina data of mitochondrial DNA sequences was pre-processed and trimmed for quality, followed by variant calling using tools including Genome Analysis Toolkit, Freebayes, and BCFtools. The variant calling analysis revealed the presence of heteroplasmy in the form of six single nucleotide polymorphisms (SNPs) within the mitochondrial genome. Specifically, three SNPs were discovered in both nad1 and nad5 genomic regions. Notably, the presence of these SNPs at high allelic frequencies strongly suggests that they are common variants rather than rare mutations. The results of the analysis of nad1 and nad5 indicate clear regions containing fixed SNPs while other regions showed only a few copies of random SNPs. This has important implications for population genetics, DNA barcoding, and phylogenetic studies that commonly utilise the cox1 gene. The presence of random SNPs (i.e., not fixed) even in small numbers, can potentially influence the interpretation of the data if such SNP-containing copies of cox1 are sequenced by chance. Although the likelihood of this is low given the vast number of non-SNP copies, it is still a factor that should be considered when concluding cox1-based analyses.

Keywords: mitochondrial DNA, molecular markers, heteroplasmy, genetic variation, bioinformatics