Publications

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.

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.Competing Interest StatementThe authors have declared no competing interest.

Schistosomes are obligately sexual blood flukes that can be maintained in the laboratory using freshwater snails as intermediate and rodents as definitive hosts. The genetic composition of laboratory schistosome populations is poorly understood: whether genetic variation has been purged due to serial inbreeding or retained is unclear. We sequenced 19 - 24 parasites from each of five laboratory Schistosoma mansoni populations and compared their genomes with published exome data from four S. mansoni field populations. We found abundant genomic variation (0.897 - 1.22 million variants) within laboratory populations: these retained on average 49% (Π ; = 3.27e-04 - 8.94e-04) of the nucleotide diversity observed in the four field parasite populations (Π ; = 1.08e-03 - 2.2e-03). However, the pattern of variation was very different in laboratory and field populations. Tajima’s D was positive in all laboratory populations except SmBRE, indicative of recent population bottlenecks, but negative in all field populations. Current effective population size estimates of laboratory populations were lower (2 - 258) compared to field populations (3,174 - infinity). The distance between markers at which linkage disequilibrium (LD) decayed to 0.5 was longer in laboratory populations (59 bp - 180 kb) compared to field populations (9 bp - 9.5 kb). SmBRE was the least variable; this parasite also shows low fitness across the lifecycle, consistent with inbreeding depression. The abundant genetic variation present in most laboratory schistosome populations has several important implications: (i) measurement of parasite phenotypes, such as drug resistance, using laboratory parasite populations will determine average values and underestimate trait variation; (ii) genome-wide association studies (GWAS) can be conducted in laboratory schistosome populations by measuring phenotypes and genotypes of individual worms; (iii) genetic drift may lead to divergence in schistosome populations maintained in different laboratories. We conclude that the abundant genetic variation retained within many laboratory schistosome populations can provide valuable, untapped opportunities for schistosome research.Competing Interest StatementThe authors have declared no competing interest.

Background: The microbiome of disease vectors can be a key determinant of their ability to transmit parasites. Conversely, parasite infection may modify vector microbiomes. We are exploring the interactions between the Biomphalaria glabrata snail microbiome and the blood fluke Schistosoma mansoni, responsible for an estimated 200,000 human deaths each year. Snail hosts vary in their susceptibility to schistosome parasites, and the underlying mechanisms driving this variation are not fully understood. We have previously shown that the snail hemolymph (i.e., blood) and organs harbor a diverse microbiome. Here we investigate the impact of schistosome infection on snail microbiomes, hypothesizing that invading schistosomes can alter the snail microbiomes in both composition and abundance over the course of infection, as developing schistosome parasites are in close contact with the host tissues. Results: We generated cohorts of uninfected and S. mansoni infected snails. We collected snail hemolymph and hepatopancreas (i.e., liver) at 8 timepoints during the pre-patent and patent periods of schistosome infection. We quantified bacterial density using qPCR and profiled the microbiome composition of all samples by sequencing the V4 region of the 16S rRNA. Schistosome infection had surprisingly no effect on bacterial density and limited effect on the microbiome composition, affecting mainly the hemolymph during the pre-patent period (at day 7 and 21). Organ and hemolymph microbiomes were relatively stable overtime for both infected and uninfected snail cohorts. The sample type (hemolymph, hepatopancreas) was the major driver of the differences observed in microbiome composition. Conclusions: The limited impact of schistosome infection on the host snail microbiomes might be explained by the long-term interaction of the two partners and the fact that parasite fitness is closely dependent on host fitness. Further investigations into the interactions between snails, their microbiomes, and schistosome parasites are essential for developing strategies to disrupt the parasite lifecycle and, consequently, schistosomiasis transmission.Competing Interest StatementThe authors have declared no competing interest.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized.

METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance.

RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted.

CONCLUSION: The malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.

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 sporocysts 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 \~10x increase in cercarial production and \~4x 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 eight-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 rose in the SmBRE population from 0 to 41-46% across the genome between September and October 2021, documenting the timing and magnitude of the contamination event. (ii) After contamination, strong selection (s = \~0.23) drove 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 fixed 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.Competing Interest StatementThe authors have declared no competing interest.

BACKGROUND: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa, and viruses, but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes.

METHODS: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over an infection period of 12 weeks. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology), and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics.

RESULTS: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area, but not in granuloma size. Variation in organ weight was explained by egg burden and by intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokines (IFN-γ, TNF-α), eosinophil, lymphocyte, and monocyte counts.

CONCLUSIONS: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotype impact immunopathology outcomes.

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.