Daily rhythms in gene expression of the human parasite Schistosoma mansoni.

BACKGROUND: The consequences of the earth's daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis. RESULTS: Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host's active phase and a 'peak in metabolic activity' during the host's resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity. CONCLUSIONS: There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.

Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection.

BACKGROUND: Chromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution. RESULTS: Long-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes. CONCLUSIONS: Despite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

Genome-wide discovery, and computational and transcriptional characterization of an AIG gene family in the freshwater snail Biomphalaria glabrata, a vector for Schistosoma mansoni.

BACKGROUND: The AIG (avrRpt2-induced gene) family of GTPases, characterized by the presence of a distinctive AIG1 domain, is mysterious in having a peculiar phylogenetic distribution, a predilection for undergoing expansion and loss, and an uncertain functional role, especially in invertebrates. AIGs are frequently represented as GIMAPs (GTPase of the immunity associated protein family), characterized by presence of the AIG1 domain along with coiled-coil domains. Here we provide an overview of the remarkably expanded AIG repertoire of the freshwater gastropod Biomphalaria glabrata, compare it with AIGs in other organisms, and detail patterns of expression in B. glabrata susceptible or resistant to infection with Schistosoma mansoni, responsible for the neglected tropical disease of intestinal schistosomiasis. RESULTS: We define the 7 conserved motifs that comprise the AIG1 domain in B. glabrata and detail its association with at least 7 other domains, indicative of functional versatility of B. glabrata AIGs. AIG genes were usually found in tandem arrays in the B. glabrata genome, suggestive of an origin by segmental gene duplication. We found 91 genes with complete AIG1 domains, including 64 GIMAPs and 27 AIG genes without coiled-coils, more than known for any other organism except Danio (with > 100). We defined expression patterns of AIG genes in 12 different B. glabrata organs and characterized whole-body AIG responses to microbial PAMPs, and of schistosome-resistant or -susceptible strains of B. glabrata to S. mansoni exposure. Biomphalaria glabrata AIG genes clustered with expansions of AIG genes from other heterobranch gastropods yet showed unique lineage-specific subclusters. Other gastropods and bivalves had separate but also diverse expansions of AIG genes, whereas cephalopods seem to lack AIG genes. CONCLUSIONS: The AIG genes of B. glabrata exhibit expansion in both numbers and potential functions, differ markedly in expression between strains varying in susceptibility to schistosomes, and are responsive to immune challenge. These features provide strong impetus to further explore the functional role of AIG genes in the defense responses of B. glabrata, including to suppress or support the development of medically relevant S. mansoni parasites.

Differential expression of small RNAs from Burkholderia thailandensis in response to varying environmental and stress conditions.

BACKGROUND: Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology. RESULTS: We profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors. In particular, the trans-encoded BTH_s1 and s39 exhibited differential expression profiles dependent on growth phase and cell stimuli, such as antibiotics and serum. Furthermore, knockdown of the highly-expressed BTH_s39 by antisense transcripts reduced B. thailandensis cell growth and attenuated host immune response upon infection, indicating that BTH_s39 functions in bacterial metabolism and adaptation to the host. In addition, expression of cis-encoded BTH_s13 and s19 found in the 5' untranslated regions of their cognate genes correlated with tight regulation of gene transcript levels. This sRNA-mediated downregulation of gene expression may be a conserved mechanism of post-transcriptional gene dosage control. CONCLUSIONS: These studies provide a broad analysis of differential Burkholderia sRNA expression profiles and illustrate the complexity of bacterial gene regulation in response to different environmental stress conditions.

The stage- and sex-specific transcriptome of the human parasite Schistosoma mansoni.

The flatworm Schistosoma mansoni is an important but neglected pathogen that causes the disease schistosomiasis in millions of people worldwide. The parasite has a complex life cycle, undergoing sexual reproduction in a mammalian host and asexual replication in a snail host. Understanding the molecular mechanisms that the parasite uses to transition between hosts and develop into dimorphic reproductively competent adults may reveal new strategies for control. We present the first comprehensive transcriptomic analysis of S. mansoni, from eggs to sexually naïve worms. Focusing on eight life stages spanning free-living water-borne and parasitic stages from both intermediate and definitive hosts, we have generated deep RNA-seq data for five replicates per group for a total of 75 data sets. The data were produced using a single approach to increase the accuracy of stage-to-stage comparisons and made accessible via a user-friendly tool to visualise and explore gene expression ( https://lifecycle.schisto.xyz/ ). These data are valuable for understanding the biology and sex-specific development of schistosomes and the interpretation of complementary genomic and functional genetics studies.

Virus-derived sequences from the transcriptomes of two snail vectors of schistosomiasis, Biomphalaria pfeifferi and Bulinus globosus from Kenya.

Schistosomiasis, which infects more than 230 million people, is vectored by freshwater snails. We identified viral sequences in the transcriptomes of Biomphalaria pfeifferi (BP) and Bulinus globosus (BuG), two of the world's most important schistosomiasis vectors in Africa. Sequences from 26 snails generated using Illumina Hi-Seq or 454 sequencing were assembled using Trinity and CAP3 and putative virus sequences were identified using a bioinformatics pipeline. Phylogenetic analyses were performed using viral RNA-dependent RNA polymerase and coat protein sequences to establish relatedness between virus sequences identified and those of known viruses. Viral sequences were identified from the entire snail holobiont, including symbionts, ingested material and organisms passively associated with the snails. Sequences derived from more than 17 different viruses were found including five near full-length genomes, most of which were small RNA viruses with positive sense RNA genomes (i.e., picorna-like viruses) and some of which are likely derived from adherent or ingested diatoms. Based on phylogenetic analysis, five of these viruses (including BPV2 and BuGV2) along with four Biomphalaria glabrata viruses reported previously, cluster with known invertebrate viruses and are putative viruses of snails. The presence of RNA sequences derived from four of these novel viruses in samples was confirmed. Identification of the genome sequences of candidate snail viruses provides a first step toward characterization of additional gastropod viruses, including from species of biomedical significance.

An Overview of Transcriptional Responses of Schistosome-Susceptible (M line) or -Resistant (BS-90) Biomphalaria glabrata Exposed or Not to Schistosoma mansoni Infection.

Background: We seek to provide a comprehensive overview of transcriptomics responses of immune-related features of the gastropod Biomphalaria glabrata (Bg) following exposure to Schistosoma mansoni (Sm), a trematode causing human schistosomiasis. Responses of schistosome-susceptible (M line, or SUS) and -resistant (BS-90, or RES) Bg strains were characterized following exposure to Sm for 0.5, 2, 8 or 40 days post-exposure (dpe). Methods: RNA-Seq and differential expression analysis were undertaken on 56 snails from 14 groups. We considered 7 response categories: 1) constitutive resistance factors; 2) constitutive susceptibility factors; 3) generalized stress responses; 4) induced resistance factors; 5) resistance factors suppressed in SUS snails; 6) suppressed/manipulated factors in SUS snails; and 7) tolerance responses in SUS snails. We also undertook a gene co-expression network analysis. Results from prior studies identifying schistosome resistance/susceptibility factors were examined relative to our findings. Results: A total of 792 million paired-end reads representing 91.2% of the estimated 31,985 genes in the Bg genome were detected and results for the 7 categories compiled and highlighted. For both RES and SUS snails, a single most supported network of genes with highly correlated expression was found. Conclusions: 1) Several constitutive differences in gene expression between SUS and RES snails were noted, the majority over-represented in RES; 2) There was little indication of a generalized stress response shared by SUS and RES snails at 0.5 or 2 dpe; 3) RES snails mounted a strong, multi-faceted response by 0.5 dpe that carried over to 2 dpe; 4) The most notable SUS responses were at 40 dpe, in snails shedding cercariae, when numerous features were either strongly down-regulated indicative of physiological distress or parasite manipulation, or up-regulated, suggestive of tolerance or survival-promoting effects; 5) Of 55 genes previously identified in genome wide mapping studies, 29 (52.7%) were responsive to Sm, as were many familiar resistance-associated genes (41.0%) identified by other means; 6) Both network analysis and remarkably specific patterns of expression of lectins and G protein-coupled receptors in categories 4, 6 and 7 were indicative of orchestrated responses of different suites of genes in SUS or RES snails following exposure to Sm.

Schistosoma mansoni Vector Snails in Antigua and Montserrat, with Snail-Related Considerations Pertinent to a Declaration of Elimination of Human Schistosomiasis.

Investigations leading to a WHO-validated declaration of elimination of schistosomiasis transmission are contemplated for several countries, including Caribbean island nations. With assistance from the Pan American Health Organization, we undertook freshwater snail surveys in two such nations, Antigua and Barbuda, and Montserrat in September and October 2017. Historically, the transmission of Schistosoma mansoni supported by the Neotropical vector snail Biomphalaria glabrata occurred in both countries. Transmission on the islands is thought to have been interrupted by the treatment of infected people, improved sanitation, introduction of competitor snails, and on Montserrat with the eruption of the Soufrière volcano which decimated known B. glabrata habitats. Guided by the available literature and local expertise, we found Biomphalaria snails in seven of 15 and one of 14 localities on Antigua and Montserrat, respectively, most of which were identified anatomically and molecularly as Biomphalaria kuhniana. Two localities on Antigua harbored B. glabrata, but no schistosome infections in snails were found. For snail-related aspects of validation of elimination, there are needs to undertake basic local training in medical malacology, be guided by historical literature and recent human schistosomiasis surveys, improve and validate sampling protocols for aquatic habitats, enlist local expertise to efficiently find potential transmission sites, use both anatomical and molecular identifications of schistosomes or putative vector snail species found, if possible determine the susceptibility of recovered Biomphalaria spp. to S. mansoni, publish survey results, and provide museum vouchers of collected snails and parasites as part of the historical record.

Transcriptional responses of Biomphalaria pfeifferi and Schistosoma mansoni following exposure to niclosamide, with evidence for a synergistic effect on snails following exposure to both stressors.

BACKGROUND: Schistosomiasis is one of the world's most common NTDs. Successful control operations often target snail vectors with the molluscicide niclosamide. Little is known about how niclosamide affects snails, including for Biomphalaria pfeifferi, the most important vector for Schistosoma mansoni in Africa. We used Illumina technology to explore how field-derived B. pfeifferi, either uninfected or harboring cercariae-producing S. mansoni sporocysts, respond to a sublethal treatment of niclosamide. This study afforded the opportunity to determine if snails respond differently to biotic or abiotic stressors, and if they reserve unique responses for when presented with both stressors in combination. We also examined how sporocysts respond when their snail host is treated with niclosamide. PRINCIPAL FINDINGS: Cercariae-producing sporocysts within snails treated with niclosamide express ~68% of the genes in the S. mansoni genome, as compared to 66% expressed by intramolluscan stages of S. mansoni in snails not treated with niclosamide. Niclosamide does not disable sporocysts nor does it seem to provoke from them distinctive responses associated with detoxifying a xenobiotic. For uninfected B. pfeifferi, niclosamide treatment alone increases expression of several features not up-regulated in infected snails including particular cytochrome p450s and heat shock proteins, glutathione-S-transferases, antimicrobial factors like LBP/BPI and protease inhibitors, and also provokes strong down regulation of proteases. Exposure of infected snails to niclosamide resulted in numerous up-regulated responses associated with apoptosis along with down-regulated ribosomal and defense functions, indicative of a distinctive, compromised state not achieved with either stimulus alone. CONCLUSIONS/SIGNIFICANCE: This study helps define the transcriptomic responses of an important and under-studied schistosome vector to S. mansoni sporocysts, to niclosamide, and to both in combination. It suggests the response of S. mansoni sporocysts to niclosamide is minimal and not reflective of a distinct repertoire of genes to handle xenobiotics while in the snail host. It also offers new insights for how niclosamide affects snails.

The in vivo transcriptome of Schistosoma mansoni in the prominent vector species Biomphalaria pfeifferi with supporting observations from Biomphalaria glabrata.

BACKGROUND: The full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used. PRINCIPAL FINDINGS: Transcripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity. CONCLUSIONS/SIGNIFICANCE: This in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated.

Transcriptomic responses of Biomphalaria pfeifferi to Schistosoma mansoni: Investigation of a neglected African snail that supports more S. mansoni transmission than any other snail species.

BACKGROUND: Biomphalaria pfeifferi is highly compatible with the widespread human-infecting blood fluke Schistosoma mansoni and transmits more cases of this parasite to people than any other snail species. For these reasons, B. pfeifferi is the world's most important vector snail for S. mansoni, yet we know relatively little at the molecular level regarding the interactions between B. pfeifferi and S. mansoni from early-stage sporocyst transformation to the development of cercariae. METHODOLOGY/PRINCIPAL FINDINGS: We sought to capture a portrait of the response of B. pfeifferi to S. mansoni as it occurs in nature by undertaking Illumina dual RNA-Seq on uninfected control B. pfeifferi and three intramolluscan developmental stages (1- and 3-days post infection and patent, cercariae-producing infections) using field-derived west Kenyan specimens. A high-quality, well-annotated de novo B. pfeifferi transcriptome was assembled from over a half billion non-S. mansoni paired-end reads. Reads associated with potential symbionts were noted. Some infected snails yielded fewer normalized S. mansoni reads and showed different patterns of transcriptional response than others, an indication that the ability of field-derived snails to support and respond to infection is variable. Alterations in transcripts associated with reproduction were noted, including for the oviposition-related hormone ovipostatin and enzymes involved in metabolism of bioactive amines like dopamine or serotonin. Shedding snails exhibited responses consistent with the need for tissue repair. Both generalized stress and immune factors immune factors (VIgLs, PGRPs, BGBPs, complement C1q-like, chitinases) exhibited complex transcriptional responses in this compatible host-parasite system. SIGNIFICANCE: This study provides for the first time a large sequence data set to help in interpreting the important vector role of the neglected snail B. pfeifferi in transmission of S. mansoni, including with an emphasis on more natural, field-derived specimens. We have identified B. pfeifferi targets particularly responsive during infection that enable further dissection of the functional role of these candidate molecules.

Corrigendum: Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

This corrects the article DOI: 10.1038/ncomms15451.

Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

Altered Gene Expression in the Schistosome-Transmitting Snail Biomphalaria glabrata following Exposure to Niclosamide, the Active Ingredient in the Widely Used Molluscicide Bayluscide.

In view of the call by the World Health Organization (WHO) for elimination of schistosomiasis as a public health problem by 2025, use of molluscicides in snail control to supplement chemotherapy-based control efforts is likely to increase in the coming years. The mechanisms of action of niclosamide, the active ingredient in the most widely used molluscicides, remain largely unknown. A better understanding of its toxicology at the molecular level will both improve our knowledge of snail biology and may offer valuable insights into the development of better chemical control methods for snails. We used a recently developed Biomphalaria glabrata oligonucleotide microarray (31K features) to investigate the effect of sublethal exposure to niclosamide on the transcriptional responses of the snail B. glabrata relative to untreated snails. Most of the genes highly upregulated following exposure of snails to niclosamide are involved in biotransformation of xenobiotics, including genes encoding cytochrome P450s (CYP), glutathione S-transferases (GST), and drug transporters, notably multi-drug resistance protein (efflux transporter) and solute linked carrier (influx transporter). Niclosamide also induced stress responses. Specifically, six heat shock protein (HSP) genes from three super-families (HSP20, HSP40 and HSP70) were upregulated. Genes encoding ADP-ribosylation factor (ARF), cAMP response element-binding protein (CREB) and coatomer, all of which are involved in vesicle trafficking in the Golgi of mammalian cells, were also upregulated. Lastly, a hemoglobin gene was downregulated, suggesting niclosamide may affect oxygen transport. Our results show that snails mount substantial responses to sublethal concentrations of niclosamide, at least some of which appear to be protective. The topic of how niclosamide's lethality at higher concentrations is determined requires further study. Given that niclosamide has also been used as an anthelmintic drug for decades and has been found to have activity against several types of cancer, our findings may be of relevance in understanding how both parasites and neoplastic cells respond to this compound.