Gene Polymorphisms Among Plasmodium vivax Geographical Isolates and the Potential as New Biomarkers for Gametocyte Detection

The unique biological features of Plasmodium vivax not only make it difficult to control but also to eliminate. For the transmission of the malaria parasite from infected human to the vector, gametocytes play a major role. The transmission potential of a malarial infection is inferred based on microscopic detection of gametocytes and molecular screening of genes in the female gametocytes. Microscopy-based detection methods could grossly underestimate the reservoirs of infection as gametocytes may occur as submicroscopic or as micro- or macro-gametocytes. The identification of genes that are highly expressed and polymorphic in male and female gametocytes is critical for monitoring changes not only in their relative proportions but also the composition of gametocyte clones contributing to transmission over time. Recent transcriptomic study revealed two distinct clusters of highly correlated genes expressed in the P. vivax gametocytes, indicating that the male and female terminal gametocytogeneses are independently regulated. However, the detective power of these genes is unclear. In this study, we compared genetic variations of 15 and 11 genes expressed, respectively, in the female and male gametocytes among P. vivax isolates from Southeast Asia, Africa, and South America. Further, we constructed phylogenetic trees to determine the resolution power and clustering patterns of gametocyte clones. As expected, Pvs25 (PVP01_0616100) and Pvs16 (PVP01_0305600) expressed in the female gametocytes were highly conserved in all geographical isolates. In contrast, genes including 6-cysteine protein Pvs230 (PVP01_0415800) and upregulated in late gametocytes ULG8 (PVP01_1452800) expressed in the female gametocytes, as well as two CPW-WPC family proteins (PVP01_1215900 and PVP01_1320100) expressed in the male gametocytes indicated considerably high nucleotide and haplotype diversity among isolates. Parasite samples expressed in male and female gametocyte genes were observed in separate phylogenetic clusters and likely represented distinct gametocyte clones. Compared to Pvs25, Pvs230 (PVP01_0415800) and a CPW-WPC family protein (PVP01_0904300) showed higher expression in a subset of Ethiopian P. vivax samples. Thus, Pvs230, ULG8, and CPW-WPC family proteins including PVP01_0904300, PVP01_1215900, and PVP01_1320100 could potentially be used as novel biomarkers for detecting both sexes of P. vivax gametocytes in low-density infections and estimating transmission reservoirs.

Extensive genetic diversity in Plasmodium vivax from Sudan and its genetic relationships with other geographical isolates

Plasmodium vivax malaria is a neglected tropical disease in Africa due to low occurrence rates and lack of accurate diagnosis. Recently, there has been a dramatic increase in P. vivax cases in East Africa and reportedly spreading to western countries. This study investigated the geographical origin and genetic diversity of P. vivax in Sudan by 14 microsatellite markers. A total of 113 clinical P. vivax samples were collected from two districts, New Halfa and Khartoum in Sudan. In addition, data from 841 geographical samples retrieved from the database for global genetic analysis were included in the analysis to further the genetic relationships among the P. vivax isolates at regional and worldwide scales. On a regional scale, we observed 91 unique and 8 shared haplotypes amongst the Sudan samples. Such a high genetic diversity compared to other geographical isolates lends support to hypothesis that P. vivax was originated from Africa. On a global scale, as already demonstrated, we observed distinct genetic clustering of P. vivax isolates from Africa, South America, and Asia (including Papua New Guinea and Solomon Island) with limited admixture in all three clusters. The principal component analysis and phylogenetic tree showed similar clustering patterns and highlighted the contribution of the African isolates to the genetic variation observed globally. The East African P. vivax showed similarity with some of the Asian isolates suggesting potential recent introductions. Our results show extensive genetic diversity co-occurring with significant multi-locus linkage disequilibrium, demonstrating the effectiveness of using microsatellite markers to implement effective control measures.

Transposable Elements are an evolutionary force shaping genomic plasticity in the parthenogenetic root-knot nematode Meloidogyne incognita

Despite reproducing without sexual recombination, the root-knot nematode Meloidogyne incognita is adaptive and versatile. Indeed, this species displays a global distribution, is able to parasitize a large range of plants and can overcome plant resistance in a few generations. The mechanisms underlying this adaptability without sex remain poorly known and only low variation at the single nucleotide polymorphism level have been observed so far across different geographical isolates with distinct ranges of compatible hosts. Hence, other mechanisms than the accumulation of point mutations are probably involved in the genomic dynamics and plasticity necessary for adaptability. Transposable elements (TEs), by their repetitive nature and mobility, can passively and actively impact the genome dynamics. This is particularly expected in polyploid hybrid genomes such as the one of M. incognita. Here, we have annotated the TE content of M. incognita, analyzed the statistical properties of this TE content, and used population genomics approach to estimate the mobility of these TEs across 12 geographical isolates, presenting phenotypic variations. The TE content is more abundant in DNA transposons and the distribution of TE copies identity to their consensuses sequence suggests they have been at least recently active. We have identified loci in the genome where the frequencies of presence of a TE showed variations across the different isolates. Compared to the M. incognita reference genome, we detected the insertion of some TEs either within genic regions or in the upstream regulatory regions. These predicted TEs insertions might thus have a functional impact. We validated by PCR the insertion of some of these TEs, confirming TE movements probably play a role in the genome plasticity with possible functional impacts.

Multilocus Sequence Typing (MLST) and Random Polymorphic DNA (RAPD) Comparisons of Geographic Isolates of Neoparamoeba perurans, the Causative Agent of Amoebic Gill Disease

Neoparamoba perurans, is the aetiological agent of amoebic gill disease (AGD), a disease that affects farmed Atlantic salmon worldwide. Multilocus sequence typing (MLST) and Random Amplified Polymorphic DNA (RAPD) are PCR-based typing methods that allow for the highly reproducible genetic analysis of population structure within microbial species. To the best of our knowledge, this study represents the first use of these typing methods applied to N. perurans with the objective of distinguishing geographical isolates. These analyses were applied to a total of 16 isolates from Australia, Canada, Ireland, Scotland, Norway, and the USA. All the samples from Australia came from farm sites on the island state of Tasmania. Genetic polymorphism among isolates was more evident from the RAPD analysis compared to the MLST that used conserved housekeeping genes. Both techniques consistently identified that isolates of N. perurans from Tasmania, Australia were more similar to each other than to the isolates from other countries. While genetic differences were identified between geographical isolates, a BURST analysis provided no evidence of a founder genotype. This suggests that emerging outbreaks of AGD are not due to rapid translocation of this important salmonid pathogen from the same area.

Cytochrome c oxidase subunit I haplotype reveals high genetic diversity of Angiostrongylus malaysiensis (Nematoda: Angiostrongylidae)

The rat lungworm Angiostrongylus malaysiensis is a metastrongyloid nematode parasite. It has been reported in Malaysia, Thailand, Laos, Myanmar, Indonesia and Japan. In this study, A. malaysiensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed high COI haplotype diversity of A. malaysiensis from Thailand. The geographical isolates of A. malaysiensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. cantonensis. In the present study, five new haplotypes were identified in addition to the four haplotypes reported in the literature. Phylogenetic analysis revealed that four of these five new haplotypes – one from Mae Hong Song (northern region), two from Tak (western region) and one from Phang Nga (southern region) – formed a distinct clade with those from Phatthalung (southern region) and Malaysia. The haplotype from Malaysia was identical to that of Phatthalung (haplotype AM1). In general, the COI sequences did not differentiate unambiguously the various geographical isolates of A. malaysiensis. This study has confirmed the presence of high COI genetic diversity in various geographical isolates of A. malaysiensis. The COI gene sequence will be suitable for studying genetic diversity, population structure and phylogeography.