Publications

There are limited control measures for the disease schistosomiasis, despite the fact that infection with parasitic blood flukes affects hundreds of millions of people worldwide. The current treatment, praziquantel, has been in use since the 1980’s and there is a concern that drug resistance may emerge with continued monotherapy. Given the need for additional antischistosomal drugs, we have re-visited an old lead, meclonazepam. In comparison to praziquantel, there has been relatively little work on its antiparasitic mechanism. Recent findings indicate that praziquantel and meclonazepam act through distinct receptors, making benzodiazepines a promising chemical series for further exploration. Previous work has profiled the transcriptional changes evoked by praziquantel treatment. Here, we examine in detail schistosome phenotypes evoked by in vitro and in vivo meclonazepam treatment. These data confirm that meclonazepam causes extensive tegument damage and directly kills parasites, as measured by pro-apoptotic caspase activation. In vivo meclonazepam exposure results in differential expression of many genes that are divergent in parasitic flatworms, as well as several gene products implicated in blood feeding and regulation of hemostasis in other parasites. Many of these transcripts are also differentially expressed with praziquantel exposure, which may reflect a common schistosome response to the two drugs. However, despite these similarities in drug response, praziquantel-resistant parasites retain susceptibility to meclonazepam’s schistocidal effects. These data provide new insight into the mechanism of antischistosomal benzodiazepines, resolving similarities and differences with the current frontline therapy, praziquantel.

Pathogen genomics is a powerful tool for tracking infectious disease transmission. In malaria, identity-by-descent is used to assess the genetic relatedness between parasites and has been used to study transmission and importation. In theory, identity-by-descent can be used to distinguish genealogical relationships to reconstruct transmission history or identify parasites for QTL experiments. MalKinID (Malaria Kinship Identifier) is a new classification model designed to identify genealogical relationships among malaria parasites based on genome-wide identity-by-descent proportions and identity-by-descent segment distributions. MalKinID was calibrated to the genomic data from 3 laboratory-based genetic crosses (yielding 440 parent-child and 9060 full-sibling comparisons). MalKinID identified lab-generated F1 progeny with >80% sensitivity and showed that 0.39 (95% CI 0.28, 0.49) of the second-generation progeny of a NF54 and NHP4026 cross were F1s and 0.56 (0.45, 0.67) were backcrosses of an F1 with the parental NF54 strain. In simulated outcrossed importations, MalKinID reconstructs genealogy history with high precision and sensitivity, with F1-scores exceeding 0.84. However, when importation involves inbreeding, such as during serial co-transmission, the precision and sensitivity of MalKinID declined, with F1-scores (the harmonic mean of precision and sensitivity) of 0.76 (0.56, 0.92) and 0.23 (0.0, 0.4) for parent-child and full-sibling and <0.05 for second-degree and third-degree relatives. Disentangling inbred relationships required adapting MalKinID to perform multisample comparisons. Genealogical inference is most powered when (1) outcrossing is the norm or (2) multisample comparisons based on a predefined pedigree are used. MalKinID lays the foundations for using identity-by-descent to track parasite transmission history and for separating progeny for quantitative-trait-locus experiments.

Background: The microbiome is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. The microbiome may influence transmission of pathogens by their vectors, such as mosquitoes or aquatic snails. We previously sequenced the V4 region of the bacterial 16S rRNA gene from the hemolymph (blood) of Biomphalaria spp. snails, vectors of the human blood fluke schistosome. We showed that snail hemolymph harbored an abundant and diverse microbiome. This microbiome is distinct from the water environment and can discriminate snail species and populations. As hemolymph bathes snail organs, we then investigated the heterogeneity of the microbiome in these organs.

Results: We dissected ten snails for each of two different species (B. alexandrina and B. glabrata) and collected their hemolymph and organs (ovotestis, hepatopancreas, gut, and stomach). We also ground in liquid nitrogen four whole snails of each species. We sampled the water in which the snails were living (environmental controls). Sequencing the 16S rRNA gene revealed organ-specific microbiomes. These microbiomes harbored a lower diversity than the hemolymph microbiome, and the whole-snail microbiome. The organ microbiomes tend to cluster by physiological function. In addition, we showed that the whole-snail microbiome is more similar to hemolymph microbiome.

Conclusions: These results are critical for future work on snail microbiomes and show the necessity of sampling individual organ microbiomes to provide a complete description of snail microbiomes.

The microbiome is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. The microbiome may influence transmission of pathogens by their vectors, such as mosquitoes or aquatic snails. We previously sequenced the V4 region of the bacterial 16S rRNA gene from the hemolymph (blood) of Biomphalaria spp. snails, vectors of the human blood fluke schistosome. We showed that snail hemolymph harbored an abundant and diverse microbiome. This microbiome is distinct from the water environment and can discriminate snail species and populations. As hemolymph bathes snail organs, we then investigated the heterogeneity of the microbiome in these organs.

We dissected ten snails for each of two different species (B. alexandrina and B. glabrata) and collected their hemolymph and organs (ovotestis, hepatopancreas, gut, and stomach). We also ground in liquid nitrogen four whole snails of each species. We sampled the water in which the snails were living (environmental controls). Sequencing the 16S rRNA gene revealed organ-specific microbiomes. These microbiomes harbored a lower diversity than the hemolymph microbiome, and the whole-snail microbiome. The organ microbiomes tend to cluster by physiological function. In addition, we showed that the whole-snail microbiome is more similar to hemolymph microbiome.

These results are critical for future work on snail microbiomes and show the necessity of sampling individual organ microbiomes to provide a complete description of snail microbiomes.

The human parasitic fluke, Schistosoma haematobium hybridizes with the livestock parasite S. bovis in the laboratory, but the frequency of hybridization in nature is unclear. Here, we analyze 34.6 million single nucleotide variants in 162 samples from 18 African countries, revealing a sharp genetic discontinuity between northern and southern S. haematobium. We find no evidence for recent hybridization. Instead the data reveal admixture events that occurred 257-879 generations ago in northern S. haematobium populations. Fifteen introgressed S. bovis genes are approaching fixation in northern S. haematobium with four genes potentially driving adaptation. Further, we identify 19 regions that are resistant to introgression; these are enriched on the sex chromosomes. These results (i) suggest strong barriers to gene flow between these species, (ii) indicate that hybridization may be less common than currently envisaged, but (iii) reveal profound genomic consequences of rare interspecific hybridization between schistosomes of medical and veterinary importance.

There are limited control measures for the disease schistosomiasis, despite the fact that infection with parasitic blood flukes affects hundreds of millions of people worldwide. The current treatment, praziquantel, has been in use since the 1980's and there is a concern that drug resistance may emerge with continued monotherapy. Given the need for additional antischistosomal drugs, we have re-visited an old lead, meclonazepam. In comparison to praziquantel, there has been relatively little work on its antiparasitic mechanism. Recent findings indicate that praziquantel and meclonazepam act through distinct receptors, making benzodiazepines a promising chemical series for further exploration. Previous work has profiled the transcriptional changes evoked by praziquantel treatment. Here, we examine in detail schistosome phenotypes evoked by in vitro and in vivo meclonazepam treatment. These data confirm that meclonazepam causes extensive tegument damage and directly kills parasites, as measured by pro-apoptotic caspase activation. In vivo meclonazepam exposure results in differential expression of many genes that are divergent in parasitic flatworms, as well as several gene products implicated in blood feeding and regulation of hemostasis in other parasites. Many of these transcripts are also differentially expressed with praziquantel exposure, which may reflect a common schistosome response to the two drugs. However, despite these similarities in drug response, praziquantel-resistant parasites retain susceptibility to meclonazepam's schistocidal effects. These data provide new insight into the mechanism of antischistosomal benzodiazepines, resolving similarities and differences with the current frontline therapy, praziquantel.

Background: Genomic analysis has revealed extensive contamination among laboratory-maintained microbes including malaria parasites, Mycobacterium tuberculosis, and Salmonella spp. Here, we provide direct evidence for recent contamination of a laboratory schistosome parasite population, and we investigate its genomic consequences. The Brazilian Schistosoma mansoni population SmBRE has several distinctive phenotypes, showing poor infectivity, reduced sporocyst number, low levels of cercarial shedding and low virulence in the intermediate snail host, and low worm burden and low fecundity in the vertebrate rodent host. In 2021 we observed a rapid change in SmBRE parasite phenotypes, with a 10-fold increase in cercarial production and fourfold increase in worm burden.

Methods: To determine the underlying genomic cause of these changes, we sequenced pools of SmBRE adults collected during parasite maintenance between 2015 and 2023. We also sequenced another parasite population (SmLE) maintained alongside SmBRE without phenotypic changes.

Results: While SmLE allele frequencies remained stable over the 8-year period, we observed sudden changes in allele frequency across the genome in SmBRE between July 2021 and February 2023, consistent with expectations of laboratory contamination. (i) SmLE-specific alleles increased in the SmBRE population from 0 to 41–46% across the genome between September and October 2021, reflecting the timing and magnitude of the contamination event. (ii) After contamination, strong selection (s ≅0.23) drove the replacement of low-fitness SmBRE with high-fitness SmLE alleles. (iii) Allele frequency changed rapidly across the whole genome, except for a region on chromosome 4, where SmBRE alleles remained at high frequency.

Conclusions: We were able to detect contamination in this case because SmBRE shows distinctive phenotypes. However, this would likely have been missed with phenotypically similar parasites. These results provide a cautionary tale about the importance of tracking the identity of parasite populations, but also showcase a simple approach to monitor changes within populations using molecular profiling of pooled population samples to characterize single-nucleotide polymorphisms. We also show that genetic drift results in continuous change even in the absence of contamination, causing parasites maintained in different labs (or sampled from the same lab at different times) to diverge.

Pathogen genomics is a powerful tool for tracking infectious disease transmission. In malaria, identity-by-descent (IBD) is used to assess the genetic relatedness between parasites and has been used to study transmission and importation. In theory, IBD can be used to distinguish genealogical relationships to reconstruct transmission history or identify parasites for quantitative-trait-locus experiments. MalKinID (Malaria Kinship Identifier) is a new classification model designed to identify genealogical relationships among malaria parasites based on genome-wide IBD proportions and IBD segment distributions. MalKinID was calibrated to the genomic data from three laboratory-based genetic crosses (yielding 440 parent-child [PC] and 9060 full-sibling [FS] comparisons). MalKinID identified lab generated F1 progeny with >80% sensitivity and showed that 0.39 (95% CI 0.28, 0.49) of the second-generation progeny of a NF54 and NHP4026 cross were F1s and 0.56 (0.45, 0.67) were backcrosses of an F1 with the parental NF54 strain. In simulated outcrossed importations, MalKinID reconstructs genealogy history with high precision and sensitivity, with F1-scores exceeding 0.84. However, when importation involves inbreeding, such as during serial co-transmission, the precision and sensitivity of MalKinID declined, with F1-scores (the harmonic mean of precision and sensitivity) of 0.76 (0.56, 0.92) and 0.23 (0.0, 0.4) for PC and FS and <0.05 for second-degree and third-degree relatives. Disentangling inbred relationships required adapting MalKinID to perform multi-sample comparisons. Genealogical inference is most powered when 1) outcrossing is the norm or 2) multi-sample comparisons based on a predefined pedigree are used. MalKinID lays the foundations for using IBD to track parasite transmission history and for separating progeny for quantitative-trait-locus experiments.

The trematodes that cause schistosomiasis in humans require aquatic snails as intermediate hosts. Identifying the genes in snails at which allelic variation controls resistance to infection by schistosomes could lead to novel ways to break the cycle of transmission. We therefore mapped genetic variation within the BS90 population of Biomphalaria glabrata snails that controls their resistance to infection by the SmLE population of Schistosoma mansoni. A marker in the PTC2 genomic region strongly associates with variation in resistance. The S-haplotype, which confers increased susceptibility, appears to be almost completely dominant to the R-haplotype, which confers increased resistance. This result suggests a model in which the parasite must match a molecule on the host side to successfully infect. The genomic region surrounding our marker shows high structural and sequence variability between haplotypes. It is also highly enriched for genes that code for single-pass transmembrane (TM1) genes. Several of the TM1 genes present on the S-haplotype lack orthologs on the R-haplotype, which makes them intriguing candidate genes in a model of dominant susceptibility. These results add to a growing body of work that suggests TM1 genes, especially those in this exceptionally diverse genomic region, may play an important role in snail-schistosome compatibility polymorphisms.

 Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.