Biology subjects

Lotkowska, M. E.

Publications and source records attributed to Lotkowska, M. E..

3 recordsLinked to original sources

bioRxiv2026

In vitro sexual dimorphism establishment in schistosomes

Schistosomes are parasitic flatworms that cause Schistosomiasis, a major Neglected Tropical Disease (NTD) that affects more than 250 million people worldwide. With two morphologically distinct sexes, a heterogametic female (ZW) and a homogametic male (ZZ), schistosomes are an exception among flatworms, which are largely hermaphroditic. Sexual dimorphism in schistosomes only becomes apparent by adulthood within the mammalian host. The cellular and molecular mechanisms underlying the sexual differentiation of these parasites are poorly understood, partly due to intrinsic challenges in assessing parasite development in vivo. Therefore, robust and reproducible approaches for maintaining and developing parasites in vitro are needed to overcome these difficulties. However, to date, only a few studies have focused on protocols that allow cultured parasites to reach sexual dimorphic stages, and none have been reproduced, limiting the ability to understand the unique sexual biology of this major human parasite. Here, we refine a protocol for long-term culture of newly transformed cercariae that developed in vitro into sexually dimorphic forms. We assessed the effect of two different sera, Foetal Bovine Serum (FBS) and Human Serum (HS), added to the culture medium supplemented with human red blood cells. We found that in contrast to FBS-cultured worms, HS-cultured parasites digested red blood cells, a crucial step for long term parasite development. Additionally, while most FBS-cultured parasites did not progress beyond an early liver stage, sexual dimorphism was clearly established in the HS-cultured worms, albeit delayed compared to in vivo development. Moreover, EdU pulse experiments revealed a continuous proliferation of cells over time in HS-cultured parasites, while a significantly lower number of proliferating cells were detected in FBS-cultured worms. This protocol paves the way to study parasite development in vitro, positively impacting the principles of the 3Rs (Replacement, Reduction and Refinement) for animal research, and allowing for in-depth studies of sexual dimorphism establishment as well as in vitro screening for novel control strategies across the life cycle of these major human parasites.

developmental biology↗

bioRxiv2020

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni

At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 ({omega}1) gene. Here, we employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.

molecular biology↗

bioRxiv2019

Population genomic evidence that human and animal infections in Africa come from the same populations of Dracunculus medinensis

BackgroundGuinea worm - Dracunculus medinensis - was historically one of the major parasites of humans and has been known since antiquity. Now, Guinea worm is on the brink of eradication, as efforts to interrupt transmission have reduced the annual burden of disease from millions of infections per year in the 1980s to only 30 human cases reported globally last year. Despite the enormous success of eradication efforts to date, one complication has arisen. Over the last few years, hundreds of dogs have been found infected with this previously apparently anthroponotic parasite, almost all in Chad. Moreover, the relative numbers of infections in humans and dogs suggests that dogs may be key in maintaining transmission in that country.\n\nResultsIn an effort to shed light on this peculiar epidemiology of Guinea worm in Chad, we have sequenced and compared the genomes of worms from dog, human and other animal infections. Confirming previous work with other molecular markers, we show that all of these worms are D. medinensis, and that the same population of worms are causing both infections, can confirm the suspected transmission between host species and detect signs of a population bottleneck due to the eradication efforts. The diversity of worms in Chad appears to exclude the possibility that there were no, or very few, worms present in the country during a 10-year absence of reported cases.\n\nConclusionsThis work reinforces the importance of adequate surveillance of both human and dog populations in the Guinea worm eradication campaign and suggests that control programs should stay aware of the possible emergence of unusual epidemiology as they approach elimination.

genomics↗