Histological sections of BS-90 snails made susceptible to <i>S. mansoni</i> infection by previous infection with irradiation-attenuated <i>E. paraensei</i>.

<p>A. Control snail not exposed to <i>S. mansoni</i> infection showing normal architecture of digestive gland. B–D. BS-90 snails with disseminated <i>S. mansoni</i> infections (arrows) following exposure to irradiated <i>E. paraensei</i>, including in the digestive gland. E. Exposure to irradiated <i>E. paraensei</i> did not result in disseminated infection or shedding of <i>E. paraensei</i> cercariae, however degenerating irradiated <i>E. paraensei</i> sporocysts were observed in the heart, as expected. Two degenerating sporocysts (arrows) of <i>E. paraensei</i> in the heart of a sensitized snail (28 dpe to <i>S. mansoni</i>). Scale bar = 10 um.</p

Knockdown of FREP3 reduces resistance to <i>S. mansoni</i> infection in BS-90 strain <i>B. glabrata</i>.

<p>A) RNAi knockdown of FREP3 in BS-90 <i>B. glabrata</i> snails confirmed at the transcriptional level by RT-PCR (25 cycles) over the course of 96 hours post injection. Shown are representative results from injection of FREP3-specific siRNA oligos and control GFP-specific siRNA oligos on FREP3 transcript expression. Experimental values are compared to the endogenous control elongation factor 1-α (EF-1α). B) Confirmation of protein-level knockdown of FREP3 in 4 individual BS-90 snails (A–D) before, and 4 days after injection of FREP3-specific siRNA oligos. FREP3 was visualized using a specific anti-FREP3 antibody. As a control for protein loading 100 µg of cell-free plasma was loaded into each well, and the same samples were probed for FREP4, using an anti-FREP4 antibody. FREP4 is a different FREP family member, related to FREP3, and was detectable in all individuals both before and after FREP3 knockdown. C) Percentage of BS-90 snails shedding <i>S. mansoni</i> cercariae (21%) after knockdown of FREP3, as compared to controls which were either susceptible M-line snails exposed to <i>S. mansoni</i> (85%) or BS-90 snails injected with the GFP siRNA constructs and challenged with <i>S. mansoni</i> (0%).</p

A. Total number of transcripts exhibiting increased (above zero line) or decreased (below zero line) expression in BS-90 snails immunocompromised by irradiated <i>E. paraensei</i> before challenge with <i>S. mansoni</i> (see key for bar colors on figure).

<p>Analysis compared experimental snails at 2 or 4 days post <i>S. mansoni</i> challenge to time and size matched control BS-90 snails exposed to irradiated <i>E. paraensei</i> only. Of the 6 individual snails analyzed at each time point, 3 were successfully infected with <i>S. mansoni</i>, and 3 remained resistant. B) Expression profiles of transcripts deemed important to <i>S. mansoni</i> resistance in snails. Fold-changes in expression of snails suppressed by irradiated <i>E. paraensei</i> before <i>S. mansoni</i> challenge are compared to snails exposed only to irradiated <i>E. paraensei</i>. Bars represent standard error (n = 3).</p

Percentage of BS-90 snails shedding <i>S. mansoni</i> cercariae after attenuation with irradiated <i>E. paraensei</i> miracidia.

<p>Experimental snails (46% infection rate) were compared to <i>S. mansoni</i>-susceptible M-line <i>B. glabrata</i> (82% infected), and to BS-90 snails challenged with <i>S. mansoni</i> only (0% infected).</p

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

<div><p>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 <i>Biomphalaria glabrata</i> oligonucleotide microarray (31K features) to investigate the effect of sublethal exposure to niclosamide on the transcriptional responses of the snail <i>B</i>. <i>glabrata</i> 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.</p></div

Hierarchical clustering of expression values from annotated genes significantly differentially expressed at all niclosamide concentrations (0.05mg/L, 0.10mg/L, and 0.15mg/L).

<p>Transcript descriptions have been reorganized by general functional categories.</p

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

<div><p>Background</p><p><i>Biomphalaria pfeifferi</i> is highly compatible with the widespread human-infecting blood fluke <i>Schistosoma mansoni</i> and transmits more cases of this parasite to people than any other snail species. For these reasons, <i>B</i>. <i>pfeifferi</i> is the world’s most important vector snail for <i>S</i>. <i>mansoni</i>, yet we know relatively little at the molecular level regarding the interactions between <i>B</i>. <i>pfeifferi and S</i>. <i>mansoni</i> from early-stage sporocyst transformation to the development of cercariae.</p><p>Methodology/Principal findings</p><p>We sought to capture a portrait of the response of <i>B</i>. <i>pfeifferi</i> to <i>S</i>. <i>mansoni</i> as it occurs in nature by undertaking Illumina dual RNA-Seq on uninfected control <i>B</i>. <i>pfeifferi</i> 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 <i>de novo B</i>. <i>pfeifferi</i> transcriptome was assembled from over a half billion non-<i>S</i>. <i>mansoni</i> paired-end reads. Reads associated with potential symbionts were noted. Some infected snails yielded fewer normalized <i>S</i>. <i>mansoni</i> 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.</p><p>Significance</p><p>This study provides for the first time a large sequence data set to help in interpreting the important vector role of the neglected snail <i>B</i>. <i>pfeifferi</i> in transmission of <i>S</i>. <i>mansoni</i>, including with an emphasis on more natural, field-derived specimens. We have identified <i>B</i>. <i>pfeifferi</i> targets particularly responsive during infection that enable further dissection of the functional role of these candidate molecules.</p></div

List of differentially expressed transcripts that have putative homologs in GenBank.

<p>Note: Genes described in the table were found to be expressed differentially in at least one concentration. If two or more concentrations resulted in differential expression, the higher or highest fold change is given in the table.</p><p>List of differentially expressed transcripts that have putative homologs in GenBank.</p

Samples used for the study with total read numbers and the percent of reads mapping to the <i>S</i>. <i>mansoni</i> genome that were filtered prior to <i>de novo</i> assemblies.

<p>Samples used for the study with total read numbers and the percent of reads mapping to the <i>S</i>. <i>mansoni</i> genome that were filtered prior to <i>de novo</i> assemblies.</p

Differential expression of <i>Biomphalaria pfeifferi</i> defense-related CDS in 1d, 3d, and shedding snails.

<p>(A) Defense CDS in the 3v3 DE analysis. (B) Pie charts of proportions of CDS found to be DE in 3v3, 3v2, and 3v1 analyses. (C) Heat maps show expression levels from each of the three DE analyses highlighting the most relevant biological functional groups. Note that the 3v3 comparison includes all 3 infected snails within a time point, whereas 3v2 includes the two infected snails with the most <i>S</i>. <i>mansoni</i> reads and the 3v1 includes only the infected snail with the fewest <i>S</i>. <i>mansoni</i> reads.</p