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The immune cells of the snail Biomphalaria glabrata are classified into hyalinocyte and granulocyte subtypes. Both subtypes are essential for the proper functioning of the snail immune response, which we understand best within the context of how it responds to challenge with the human parasite Schistosoma mansoni. Granulocytes are adherent phagocytic cells that possess conspicuous granules within the cell cytoplasm. Hyalinocytes, on the other hand, are predominantly non-adherent and are known to produce a handful of anti-S. mansoni immune effectors. While our understanding of these cells has progressed, an in-depth comparison of the functional capabilities of each type of immune cell has yet to be undertaken. Here, we present the results of a single-cell RNA-seq study in which single granulocytes and hyalinocytes from S. mansoni-susceptible M-line B. glabrata and S. mansoni-resistant BS-90 B. glabrata are compared without immune stimulation. This transcriptomic analysis supports a role for the hyalinocytes as producers of immune effectors such as biomphalysin and thioester-containing proteins. It suggests that granulocytes are primarily responsible for producing fibrinogen-related proteins and are armed with various pattern-recognition receptors such as toll-like receptors with a confirmed role in the anti-S. mansoni immune response. This analysis also confirms that the granulocytes and hyalinocytes of BS-90 snails are generally more immunologically prepared than their M-line counterparts. As the first single-cell analysis of the transcriptional profiles of B. glabrata immune cells, this study provides crucial context for understanding the B. glabrata immune response. It sets the stage for future investigations into how each immune cell subtype differs in its response to various immunological threats.

We provide the first documented case of whirling disease (WD) impacts to wild, self-sustaining rainbow trout (RNTR, Oncorynchus mykiss) populations in Canada. Myxobolus cerebralis (Mc), the causative agent of WD, was first confirmed in Alberta in 2016. However, evidence of disease in local fish populations was unknown. Using a weight-of-evidence approach, we examined multiple parasite life cycle stages in the Crowsnest River, Alberta. Percentage of infected Tubifex tubifex worms actively shedding triactinomyxons (TAMs) exceeded known thresholds of Mc establishment and TAM densities instream exceeded thresholds known to cause ≥90% declines in RNTR populations. Mc was detected at 5 of 6 study sites in water, fish, and worms. Disease severity was highest in the lower watershed where 100% of sentinel fish tested positive for Mc 7 to 14 days post-exposure; up to 85% of wild fingerling RNTR showed clinical signs of disease and yearling trout were largely absent from the river suggesting reduced survival. Our findings indicate conditions necessary for outbreak of WD exist in Alberta, highlighting the need to consider this disease as an emerging threat to wild salmonid populations.

Schistosoma mansoniemploys immune evasion and immunosuppression to overcome immune responses mounted by its snail and human hosts. Myriad immunomodulating factors underlie this process, some of which are proteases. Here, we demonstrate that one protease, an invadolysin we have termed SmCI-1, is released from the acetabular glands ofS. mansonicercaria and is involved in creating an immunological milieu favorable for survival of the parasite. We confirm the presence of SmCI-1 in the cercarial stage ofS. mansoniand demonstrate that it is released during transformation into the schistosomula. We confirm SmCI-1 functions as a metalloprotease with the capacity to cleave collagen type IV, gelatin and fibrinogen. We also show that complement component C3b is cleaved by this protease, resulting in inhibition of the classical and alternative complement pathways. Finally, we assess the effect of SmCI-1 on cytokine release from human peripheral blood mononuclear cells, providing compelling evidence that SmCI-1 promotes an anti-inflammatory microenvironment by enhancing production of IL-10 and suppressing the production of inflammatory cytokines like IL-1B and IL-12p70 and those involved in eosinophil recruitment and activation, like eotaxin-1 and IL-5.

Schistosoma mansoni employs immune evasion and immunosuppression to overcome immune responses mounted by its snail and human hosts. Myriad immunomodulating factors underlie this process, some of which are proteases. Here, we demonstrate that one protease, an invadolysin we have termed SmCI-1, is released from the acetabular glands of S. mansoni cercaria and is involved in creating an immunological milieu favorable for survival of the parasite. The presence of SmCI-1 in the cercarial stage of S. mansoni is released during transformation into the schistosomula. SmCI-1 functions as a metalloprotease with the capacity to cleave collagen type IV, gelatin and fibrinogen. Additionally, complement component C3b is cleaved by this protease, resulting in inhibition of the classical and alternative complement pathways. Using SmCI-1 knockdown cercariae, we demonstrate that SmCI-1 protects schistosomula from complement-mediated lysis in human plasma. We also assess the effect of SmCI-1 on cytokine release from human peripheral blood mononuclear cells, providing compelling evidence that SmCI-1 promotes an anti-inflammatory microenvironment by enhancing production of IL-10 and suppressing the production of inflammatory cytokines like IL-1B and IL-12p70 and those involved in eosinophil recruitment and activation, like Eotaxin-1 and IL-5. Finally, we utilize the SmCI-1 knockdown cercaria in a mouse model of infection, revealing a role for SmCI-1 in S. mansoni survival.

To control swimmer's itch in northern Michigan inland lakes, USA, one species of bird, the common merganser (Mergus merganser), has been relocated from several lakes since 2015. Relocation efforts are driven by a desire to reduce the prevalence of the swimmer's itch-causing parasite Trichobilharzia stagnicolae. The intention of this state-sponsored control effort was to interrupt the life cycle of T. stagnicolae and reduce parasite egg contribution into the environment from summer resident mergansers such that infections of the intermediate snail host Stagnicola emarginata declined. Reduced snail infection prevalence was expected to substantially reduce the abundance of the swimmer's itch-causing cercarial stage of the parasite in water. With no official programme in place to assess the success of this relocation effort, we sought to study the effectiveness and impact of the removal of a single definitive host from a location with high definitive host and parasite diversity. This was assessed through a comprehensive, lake-wide monitoring study measuring longitudinal changes in the abundance of three species of avian schistosome cercariae in four inland Michigan lakes. Environmental measurements were also taken at these lakes to understand how they can affect swimmer's itch incidence. This study demonstrates that the diversity of avian schistosomes at the study lakes would likely make targeting a single species of swimmer's itch-causing parasite meaningless from a swimmer's itch control perspective. Our data also suggest that removing the common merganser is not an effective control strategy for the T. stagnicolae parasite, likely due to contributions of the parasite made by non-resident birds, possibly migrants, in the autumn and spring. It appears likely that only minimal contact time between the definitive host and the lake ecosystem is required to contribute sufficient parasite numbers to maintain a thriving population of parasite species with high host specificity.

To control swimmer’s itch in northern Michigan inland lakes, one species of bird, the common merganser (Mergus merganser), has been relocated from several lakes since 2015. Relocation efforts are driven by a desire to reduce the prevalence of the swimmer’s itch-causing parasite Trichobilharzia stagnicolae. The intention of this state-sponsored control effort was to interrupt the life cycle of T. stagnicolae and reduce parasite egg contribution into the environment from summer resident mergansers such that infections of the intermediate snail host Stagnicola emarginata declined. Reduced snail infection prevalence was expected to greatly reduce abundance of the swimmer’s itch-causing cercarial stage of the parasite in water. With no official program in place to assess the success of this relocation effort, we sought to study the effectiveness and impact of the removal of a single definitive host from a location with high definitive host and parasite diversity. This was assessed through a comprehensive, lake-wide monitoring study measuring longitudinal changes in the abundance of three species of avian schistosome cercariae in four inland Michigan lakes. Environmental measurements were also taken at these lakes to understand how they can affect swimmer’s itch incidence. Results from this study demonstrate that the diversity of avian schistosomes at the study lakes would likely make targeting of a single species of swimmer’s itch-causing parasite meaningless from a swimmer’s itch control perspective. Our data also suggest that removal of the common merganser is not an effective control strategy for the T. stagnicolae parasite, likely due to parasite contributions of migratory birds in the fall and spring. This suggests that only minimal contact time between the definitive host and the lake ecosystem is required to contribute sufficient parasite numbers to maintain a thriving population of parasite species with a high host-specificity.

Myxobolus cerebralis is the causative agent of whirling disease in salmonid fishes. In 2016, this invasive parasite was detected in Alberta, Canada, for the first time, initiating a comprehensive 3 yr monitoring program to assess where the parasite had spread within the province. As part of this program, a qPCR-based test was developed to facilitate detection of the environmental stages of M. cerebralis and from the oligochaete host, Tubifex tubifex. During this program, ~1500 environmental samples were collected and tested over 3 yr. Fish were collected from the same watersheds over 2 yr and tested as part of the official provincial monitoring effort. Substrate testing identified sites positive for M. cerebralis in 3 of 6 watersheds that had been confirmed positive by fish-based testing and 3 novel detections where the parasite had not been detected previously. Testing of individually isolated Tubifex from each sample site was used to further confirm the presence of M. cerebralis. DNA barcoding of the cytochrome oxidase I (cox1) gene of 567 oligochaete specimens collected from 6 different watersheds yielded 158 unique sequences belonging to 21 genera and 37 putative species. Phylogenetic analyses of sequences assigned to the genus Tubifex predicted 5 species of Tubifex arising from this assessment. Based on our results, we propose that environmental and worm samples can be a valuable complement to the gold-standard fish testing and will be especially useful for monitoring in areas where fish collection is challenging or prohibitive because of site accessibility or vulnerability of the fish populations.

Swimmer's itch has historically been controlled by applying copper sulfate (CuSO4) to lakes as a way to eliminate snails that serve as the intermediate hosts for swimmer's itch-causing parasites. CuSO4 is still sometimes applied specifically to areas of lakes where swimmer's itch severity is high. It is unclear whether targeted application of chemical molluscicides like CuSO4 is effective for controlling swimmer's itch. Previous research has found that the larval stage of the parasites responsible for swimmer's itch are released from infected snails and are concentrated by onshore and alongshore winds, and thus, may not be affected by such focused applications. In this study, we evaluated the impact of targeted CuSO4 application to a specific recreational swimming area in a lake in Michigan. We measured the effect on snail populations, as well as on the presence/abundance of swimmer's itch-causing parasites using qPCR. Ultimately, while CuSO4 was confirmed to significantly reduce populations of snails within the treatment area, it was found to have no significant impact on swimmer's itch-causing parasites in the water, likely due to the free-swimming larval stages (cercariae) moving into the treatment area from surrounding regions.

Freshwater gastropods of the genera Lymnaea Lamarck, 1799, Physa Draparnaud, 1801, Gyraulus Charpentier, 1837, Radix Montfort, 1810, and Stagnicola Jeffreys, 1830 are considered suitable intermediate hosts for avian schistosomes. A large trematode biodiversity survey performed across 3 yr on 6 lakes in Alberta confirmed 3 already-reported snail hosts for 7 North American avian schistosomes; however, the cytochrome c oxidase subunit 1 (COI) nucleotide sequence from 1 cercarial sample (from a single specimen of Planorbella trivolvis) was distinct from all other COI schistosome sequences. As part of a simultaneous, comparable study of P. trivolvis by us in Michigan, we collected another cercarial type from 6 lakes that was 99% similar (COI) to the aforementioned cercarial type. Phylogenetic analyses of the COI and 28S rDNA genes recovered the former cercaria in a clade of avian schistosomes. In Michigan, the feces of a Canada goose (Branta canadensis Linnaeus, 1758) had a miracidium with an identical COI nucleotide sequence. Preliminary swimmer's itch and cercarial emergence studies were performed to determine if the cercariae could cause swimmer's itch and to study the emergence pattern as compared with species of Trichobilharzia Skrjabin and Zakharow, 1920.

Myxobolus cerebralis is the causative agent of whirling disease in salmonid fishes. In 2016, this invasive parasite was detected in Alberta, Canada, for the first time, initiating a comprehensive 3 yr monitoring program to assess where the parasite had spread within the province. As part of this program, a qPCR-based test was developed to facilitate detection of the environmental stages of M. cerebralis and from the oligochaete host, Tubifex tubifex. During this program, ~1500 environmental samples were collected and tested over 3 yr. Fish were collected from the same watersheds over 2 yr and tested as part of the official provincial monitoring effort. Substrate testing identified sites positive for M. cerebralis in 3 of 6 watersheds that had been confirmed positive by fish-based testing and 3 novel detections where the parasite had not been detected previously. Testing of individually isolated Tubifex from each sample site was used to further confirm the presence of M. cerebralis. DNA barcoding of the cytochrome oxidase I (cox1) gene of 567 oligochaete specimens collected from 6 different watersheds yielded 158 unique sequences belonging to 21 genera and 37 putative species. Phylogenetic analyses of sequences assigned to the genus Tubifex predicted 5 species of Tubifex arising from this assessment. Based on our results, we propose that environmental and worm samples can be a valuable complement to the gold-standard fish testing and will be especially useful for monitoring in areas where fish collection is challenging or prohibitive because of site accessibility or vulnerability of the fish populations.

Background: Marine molluscs living in intertidal and estuarine areas, such as oysters, face numerous pathogen challenges during their development. Infection from bacteria such as Vibrio alginolyticus, represents a major factor affecting larval development and frequently leads to high mortality of the pacific oyster, Crassostrea gigas. The oyster immune response is known to play an important role in protecting the animal during development by mitigating the consequences of infection. Results: In this study, we undertook a comprehensive analysis of the immune response of C. gigas to V. alginolyticus challenge. We sequenced the transcriptome of C. gigas at 0, 6, 12, 24, 48 and 72 hours post infection. After RNA-seq, the raw reads are available through the NCBI Sequence Read Archive under accession number PRJNA623063. After filtering, a total of 58.24 Gb clean reads were produced and assembled using the reference genome of C. gigas. The distribution of quality Q30 was higher than 90.88% for each sample and the GC content ranged from 41.27% to 42.91%. When compared with sequences in the COG, GO, KEGG, Swiss-Prot, and NR databases, there were 1267, 1112, 2187, 682, 1133 differentially expressed genes annotated at 6, 12, 24, 48, 72 hours post infection respectively. Numerous immune-related genes displayed differential expression that varied over time: toll-like receptors, tripartite motif proteins, Lectin-like factors, scavenger receptors, signaling pathway components such as Myeloid differentiation factor 88, and stress proteins such as Heat shock 70 kDa protein were all found to be higher in abundance following V. alginolyticus challenge compared to control. For analysis, these genes were divided into several categories such as pattern recognition receptors, fibrinogen-like proteins, damage associated molecular patterns, complement factors, etc. These general categories allowed us to generate an immune response profile for C. gigas over the first 72 hours of infection. These results indicate that bacterial infection induce a complex pattern of immune gene expression in C. gigas larvae. Conclusion: Our study will facilitate targeted investigation into the function of specific immune factors that may explain the diversity and evolution of invertebrate immune molecules and lead to the development of effective measures to improve the performance of oyster culture.

Recreational water monitoring can be challenging due to the highly variable nature of pathogens and indicator concentrations, the myriad of potential biological hazards to measure for, and numerous access points, both official and unofficial, that are used for recreation. The aim of this study was to develop, deploy, and assess the effectiveness of a quantitative polymerase chain reaction (qPCR) community-based monitoring (CBM) program for the assessment of bacterial and parasitic hazards in recreational water. This study developed methodologies for performing qPCR ‘in the field’, then engaged with water management and monitoring groups, and tested the method in a real-world implementation study to evaluate the accuracy of CBM using qPCR both quantitatively and qualitatively. This study found high reproducibility between qPCR results performed by non-expert field users and expert laboratory results, suggesting that qPCR as a methodology could be amenable to a CBM program.

Gastropod molluscs, which have co-evolved with parasitic digenean trematodes for millions of years, utilize circulating heamocytes as the primary method of containing and killing these invading parasites. In order to do so, they must generate suitable amounts of haemocytes that are properly armed to kill parasitic worms. One method by which they generate the haemocytes required to initiate the appropriate cell mediated immune response is via the production and post-translational processing of granulins. Granulins are an evolutionarily conserved family of growth factors present in the majority of eukaryotic life forms. In their pro-granulin form, they can elicit cellular replication and differentiation. The pro-granulins can be further processed by elastase to generate smaller granulin fragments that have been shown to functionally differ from the pro-granulin precursor. In this study, we demonstrate that in vivo addition of Biomphalaria glabrata pro-granulin (BgGRN) can reduce Schistosoma mansoni infection success in numerous Biomphalaria sp. when challenged with different S. mansoni strains. We also demonstrate that cleavage of BgGRN into individual granulin subunits by elastase results in the stimulation of haemocytes to produce reactive oxygen species.

Swimmer’s itch (cercarial dermatitis) is a nuisance encountered by bathers and recreational water users worldwide. The condition is caused by the penetration of larval digenean trematodes (cercariae) of the family Schistosomatidae, into the skin, following their release into freshwater from pulmonate snails that serve as the intermediate hosts for these parasites. This study utilizes qPCR-based cercariometry to monitor and quantify cercariae from water samples collected at 5 lakes in northern Michigan. The resolution provided by qPCR facilitated assessment of the environmental and biological drivers of swimmer’s itch-causing cercariae concentrations, allowing us to demonstrate that cercarial abundance is greatest at the top of the water column, in locations with prevailing on- and alongshore winds. Electronic supplementary material The online version of this article (10.1007/s10393-018-1362-1) contains supplementary material, which is available to authorized users.

Immune factors in snails of the genusBiomphalariaare critical for combatingSchistosoma mansoni, the predominant cause of human intestinal schistosomiasis. Independently, many of these factors are known to play an important role in, but not fully define, the compatibility between the model snailB. glabrata,andS. mansoni. Here, we demonstrate association between four, previously characterized humoral immune molecules;BgFREP3,BgTEP1,BgFREP2 and Biomphalysin. We also identify unique immune determinants in the plasma ofS. mansoni-resistantB. glabratathat explain the incompatible phenotype. These factors coordinate to initiate haemocyte-mediated deestruction ofS. mansonisporocysts via production of reactive oxygen species. The inclusion ofBgFREP2 in aBgFREP3-initiated complex that also includesBgTEP1 almost completely explains resistance toS. mansoniin this model. Our study unifies many independent lines of investigation to provide a more comprehensive understanding of the snail immune system in the context of infection by this important human parasite.

Swimmer's itch is an allergic condition that occurs when the motile and infectious stage of avian schistosomes penetrate the skin of an individual. Flatworm parasites that cause swimmer's itch belong to the family Schistosomatidae. They utilize a variety of different species of bird and mammal as definitive hosts, and rely on different species of snail, in which they complete their larval development to culminate in a motile, aquatic, infectious stage called a cercaria. Recently, qPCR-based assays have been developed to monitor for swimmer's itch-causing trematodes in recreational water. This environmental DNA approach has been useful for quantifying the abundance of the free-living cercaria, the causative agent of swimmer's itch. However, the existing qPCR test amplifies from all known schistosome species, making it excellent for assessing a site for swimmer's itch potential, but not useful in determining the specific species contributing to swimmer's itch or the likely hosts (snail and bird) of the swimmer's itch-causing parasites. Thus, species-specific resolution built into a qPCR test would be useful in answering ecological questions about swimmer's itch cause, and efficacy of control efforts. This paper details bird, snail, and cercaria surveys conducted in the summer of 2018, that culminated in the development and deployment of four species-specific qPCR assays, capable of detecting Trichobilharzia stagnicolae, Trichobilharzia szidati, Trichobilharzia physellae, and Anserobilharzia brantae in recreational water. These assays were used to assess the relative abundance of each parasite in water samples collected from lakes in Northern Michigan., Graphical abstract Image 1, Highlights • qPCR diagnostic tests of four species of avian schistosomes. • Comprehensive snail, bird, and cercariae surveys in Northern Michigan. • Use of qPCR cercariometry in water to measure swimmer's itch.

Swimmer's itch has historically been controlled by applying copper sulfate (CuSO

Haematopoiesis is a process that is responsible for generating sufficient numbers of blood cells in the circulation and in tissues. It is central to maintenance of homeostasis within an animal, and is critical for defense against infection. While haematopoiesis is common to all animals possessing a circulatory system, the specific mechanisms and ultimate products of haematopoietic events vary greatly. Our understanding of this process in non-vertebrate organisms is primarily derived from those species that serve as developmental and immunological models, with sparse investigations having been carried out in other organisms spanning the metazoa. As research into the regulation of immune and blood cell development advances, we have begun to gain insight into haematopoietic events in a wider array of animals, including the molluscs. What began in the early 1900’s as observational studies on the morphological characteristics of circulating immune cells has now advanced to mechanistic investigations of the cytokines, growth factors, receptors, signalling pathways, and patterns of gene expression that regulate molluscan haemocyte development. Emerging is a picture of an incredible diversity of developmental processes and outcomes that parallels the biological diversity observed within the different classes of the phylum Mollusca. However, our understanding of haematopoiesis in molluscs stems primarily from the three most-studied classes, the Gastropoda, Cephalopoda and Bivalvia. While these represent perhaps the molluscs of greatest economic and medical importance, the fact that our information is limited to only 3 of the 9 extant classes in the phylum highlights the need for further investigation in this area. In this review, we summarize the existing literature that defines haematopoiesis and its products in gastropods, cephalopods and bivalves.

Background Cercarial dermatitis, colloquially “swimmer’s itch”, is a rash contracted in natural bodies of water, when people are exposed to skin-penetrating, larval flatworm parasites of the family Schistosomatidae, that emerge from aquatic snails. Swimmer’s itch is a globally-distributed, allergic condition, of which we know very little regarding local dynamics of transmission. This study aims to gather relevant information on swimmer’s itch in Canada, from multiple perspectives, including the human experience, parasite and host presence and distributions, and insight from historical perspectives. Methods Herein we utilize a mixed-methods approach towards examining the environmental health issue of swimmer’s itch in Canadian lakes from a nation-wide viewpoint, with an example from Alberta. We examine the human perspective of having contracted swimmer’s itch through a self-reporting surveillance system implemented over a 5-year period. We also conducted a 3-year species survey of parasites and intermediate snail hosts within lakes in central Alberta and compiled this data with snail and vertebrate (definitive) host survey data from across Alberta to examine potential for future spread. We compare the results from our surveys to a historical review of the literature to examine the extent of swimmer’s itch across Canada and identify where future efforts should be focused. Results Over 3800 cases of swimmer’s itch were captured across Canada by the self-reporting surveillance system. Swimmer’s itch cases were reported from every province except Prince Edward Island. Species surveys in Alberta revealed 7 new parasite and host records, with potential for swimmer’s itch to occur throughout most of the province based on host distributions. A review and comparison to the literature has highlighted several knowledge gaps surrounding schistosome species, host species and their distributions and contributions towards swimmer’s itch. Conclusions Swimmer’s itch is a greater environmental health hazard across Canada than previous literature would have alluded. This study provides proof-of-concept for the utility of a self-reporting surveillance system for swimmer’s itch in Canada. Recommendations are made towards implementing a systems-thinking approach that incorporates citizen-scientists for future research, management, and policy surrounding swimmer’s itch. Electronic supplementary material The online version of this article (10.1186/s12940-018-0417-7) contains supplementary material, which is available to authorized users.

Globally over 200 million people are infected with schistosomiasis, and approximately 80% are caused by just two of five species, Schistosoma haematobium and Schitosoma mansoni that are broadly distributed, and often overlap across sub-Saharan Africa. Like most neglected tropical diseases, mortality is low (an estimated 200,000 deaths annually) and morbidity is considerably high and probably underestimated. Surprisingly, little attention has been given to co-infection with these two species. We have studied co-infection with S. mansoni and S. haematobium in a peri-urban community in Ghana, one of the most highly endemic countries for schistosomiasis. We collected and examined snails of the two intermediate host species from the reservoir adjacent to the community. We also used microscopical examination of stool and urine samples to determine the level of concurrent S. mansoni and S. haematobium infections in school and administered questionnaires to assess water contact activities that predispose pupils to infections Examination of the snail hosts revealed that 0.7% (7/896) of Bulinus truncatus and 1.7% (14/780) of Biomphalaria pfeifferi snails were found to be hosting cercariae morphologically consistent with that of S. haematobium and S. mansoni respectively. The overall prevalence values for urogenital and intestinal schistosomiasis were 66.8% (135/202) and 90.1% (163/181) respectively. Only 50 of 181 schistosome-infected pupils had single-species infections and the remaining 131 pupils presented concurrent infections. Among the 131 infected with both species were 50 individuals having only S. mansoni eggs in stool and S. haematobium eggs in urine (conventional presentation). Eighty-one children (81) had eggs of both species in either urine and/or stool (ectopic presentation). From these 81, 63 had eggs of both species in urine, 6 had both species in stool, and 12 had eggs of both species present in both urine and stool. A comparatively large number of individuals from the concurrent infected group presented high and moderate infection intensities than the single infected groups. The overwhelmingly high prevalence of concurrent infections indicates further study of co-infection is needed, and points to a need call for a holistic disease control plan so Ghana can be part of nations to achieve the WHO roadmap target for schistosomiasis control by 2020.

Fibrinogen-related proteins or FREPs constitute a large family of molecules, defined by the presence of a fibrinogen-related domain (FReD). These molecules are found in all animals and are diverse in both form and function. Here, we review the current understanding of gastropod FREPs, which are characterized by the presence of a fibrinogen domain connected to one or two immunoglobulin superfamily domains by way of a short interceding region. We present a historical perspective on the discovery of FREPs in gastropods followed by a summary of advances made in the nearly two decades of research focused on the characterization of FREPs in Biomphalaria glabrata (BgFREPs). Topics covered include BgFREP genomic architecture, predicted structure and known functions, structural comparisons between BgFREPs, and evidence of somatic diversification. Also examined are the expression patterns of BgFREPs during snail development and immunological challenges. Recent functional characterization of the role BgFREPs play in the defence response against digenean trematodes is also presented, as well as new data investigating the nucleotide-level genomic conservation of FREPs among Pulmonate gastropods. Finally, we identify areas in need of further research. These include confirming and identifying the specific binding targets of BgFREPs and elucidating how they later engage snail haemocytes to elicit an immunological response, precise mechanisms and importance of BgFREP diversification, characterizing the tissue expression patterns of BgFREPs, as well as addressing whether gastropod FREPs retain immunological importance in alternative snail-trematode associations or more broadly in snail-pathogen interactions.

The colony-stimulating factor-1 (CSF-1) is the principal regulator of the survival, proliferation, differentiation, and function of macrophages and their precursors, and has been shown to play a role in the etiology of inflammation. We recently identified a novel mechanism for the control of CSF-1 activity in teleost fish, through the production of an inhibitory soluble form of the CSF-1 receptor (sCSF-1R). Primary goldfish kidney macrophages selectively expressed sCSF-1R during the senescence phase, which corresponds to a defined stage of in vitro culture development where inhibition of macrophage proliferation and apoptotic cell death are prominent. In contrast, primary macrophage cultures undergoing active proliferation displayed low levels of sCSF-1R expression. Addition of purified recombinant sCSF-1R to developing primary macrophage cultures leads to a dose-dependent decrease in macrophage proliferation and inhibits macrophage antimicrobial functions including chemotaxis, phagocytosis, and production of reactive oxygen intermediates. Using a goldfish in vivo model of self-resolving peritonitis, we found that sCSF-1R plays a role in the inhibition of inflammation, following an initial acute phase of antimicrobial responses within an inflammatory site. Soluble CSF-1R inhibits pro-inflammatory cytokine production, inhibits leukocyte recruitment to the inflammatory site and decreases ROS production in a dose-dependent manner. This sCSF-1R-dependent regulation of inflammation appears to be an elegant mechanism for the control of macrophage numbers at inflammatory sites of lower vertebrates. Overall, our results provide new insights into the evolutionary origins of the CSF-1 immune regulatory axis.

The snail's immune response is an important determinant of schistosome infection success, acting in concert with host, parasite, and environmental factors. Coordinated by haemocytes and humoral factors, it possesses immunological hallmarks such as pattern recognition receptors, and predicted gastropod-unique factors like the immunoglobulin superfamily domain-containing fibrinogen-related proteins. Investigations into mechanisms that underpin snail-schistosome compatibility have advanced quickly, contributing functional insight to many observational studies. While the snail's immune response is important to continue studying from the perspective of evolutionary immunology, as the foundational determinants of snail-schistosome compatibility continue to be discovered, the possibility of exploiting the snail for schistosomiasis control moves closer into reach. Here, we review the current understanding of immune mechanisms that influence compatibility between Schistosoma mansoni and Biomphalaria glabrata.

Significant effort has gone into assessing the fate and removal of viruses, bacteria, and protozoan parasites during wastewater treatment to provide data addressing potential health risks associated with reuse options. Comparatively less is known about the fate of parasitic worm species ova in these complex systems. It is largely assumed that these helminths settle, are removed with the sludge, and consequently represent a relatively low risk for wastewater reuse applications. However, helminths are a highly diverse group of organisms that display a wide range of physical properties that complicate the application of a single treatment for helminth reduction during wastewater treatment. Moreover, their diverse biological and physical properties make some ova highly resistant to both disinfection (i.e., with chlorine or UV treatment) and physical removal (settling) through the wastewater treatment train, indicating that there may be reason to broaden the scope of our investigations into whether parasitic worm eggs can be identified in treated wastewater. The ubiquitous human parasitic nematode Enterobius vermicularis (pinworm) produces small, buoyant ova. Utilizing a novel diagnostic quantitative PCR (qPCR), this study monitored E. vermicularis presence at two full-scale wastewater treatment plants over the course of 8 months and demonstrated incomplete physical removal of E. vermicularis ova through tertiary treatment, with removal efficiencies approximating only 0.5 and 1.6 log 10 at the two wastewater treatment plants based on qPCR. These findings demonstrate the need for more-diverse surrogates of helminthic ova to fully assess treatment performance with respect to reclaimed wastewaters. IMPORTANCE Helminths, despite being a diverse and environmentally resistant class of pathogens, are often underestimated and ignored when treatment performance at modern wastewater treatment plants is considered. A one-size-fits-all surrogate for removal of helminth ova may be inappropriate to adequately assess risk and ensure public safety when treated and partially treated wastewaters are encountered. This study argues for the use of human pinworm as a conservative indicator of the presence of helminth ova due to its small size, buoyancy, prevalence in humans, and environmental resistance.

Zygocercous (aggregating) cercarial larvae were recently discovered emerging from a physid snail during a molecular survey of cercariae from molluscs in lakes in central Alberta, Canada. This manuscript delves into the characterization of these cercariae through morphological and molecular techniques and provides the first genetic information for a zygocercous larval trematode. Analyses of cytochrome c oxidase I of mitochondrial DNA and two partial regions of nuclear ribosomal DNA sequences revealed the zygocercous cercariae to belong to the genus Australapatemon Sudarikov, 1959. Further analyses of sequences of Australapatemon burti (Miller, 1923), from cercariae and adults collected from across North America, indicate a complex of nine genetically-distinct lineages within this species, a surprising level of diversity. The zygocercous cercariae, along with adult worms collected from ducks in Manitoba, Canada, and from Mexico, represent one of these lineages, and are herein described as Australapatemon mclaughlini n. sp. Seven lineages cannot yet be identified, but one is tentatively identified as Australapatemon burti.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis., Author summary Schistosomiasis is one of the most prevalent parasitic diseases in the world. Though treatments for schistosomiasis infection exist, there is no vaccine, and reinfection is common in areas where the parasite occurs. One possible way to mitigate schistosomiasis is by controlling the transmission of the parasite larvae from the snails that carry them. Understanding the snail-parasite relationship is essential for the development of new means to interrupt transmission of the parasite from snails to humans. Snails possess immune mechanisms for fighting infection, most of which are based in hemolymph tissue. Here we characterize a novel protein, Grctm6, in a snail host of schistosome parasites. Grctm6 is structurally similar to certain other immune proteins and is present in snail hemolymph. Importantly, we demonstrate that the release of schistosomes by infected snails is exacerbated when this protein is experimentally suppressed in live snails. These results support the suitability of Grctm6 as a possible target for reducing the transmission of this human disease.

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., Biomphalaria glabrata is a fresh water snail that acts as a host for trematode Schistosoma mansoni that causes intestinal infection in human. This work describes the genome and transcriptome analyses from 12 different tissues of B glabrata, and identify genes for snail behavior and evolution.

Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection.

Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to β-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which shed S. mansoni cercariae 1-week before the susceptible controls. Our results represent the first functional characterization of a gastropod TLR, and demonstrate that BgTLR is an important snail immune receptor that is capable of influencing infection outcome following S. mansoni challenge., Author Summary The freshwater snail Biomphalaria glabrata is the subject of intensive research, primarily due to its biomedical importance as an intermediate host for the parasitic flatworm Schistosoma mansoni, which is a causative agent of the disease schistosomiasis–a chronic, debilitating condition that affects over 260 million people worldwide. Studies of this snail have led to the identification of many factors, some with high homology to known immune molecules in other organisms and many more that are unique with no known homology. However, research into the functional and mechanistic roles of these factors has only recently progressed. In this study, we have demonstrated the functional relevance of a Toll-like receptor (TLR) that we identified in B. glabrata. Transcriptional expression of this TLR was rapidly induced in a snail strain that is resistant to S. mansoni, in a pattern that is consistent with their phenotype. Furthermore, knockdown of the TLR resulted in 43% of resistant snails becoming infected with S. mansoni to patency. This advances our understanding of the mechanistic basis of snail-schistosome compatibility significantly, and may one day facilitate the development of tools for improving the control of schistosomiasis. It also contributes novel functionality to TLRs, one of the most evolutionarily conserved pattern-recognition receptors and cognate signalling pathways in immunity.

Digenean trematode distributions, compatibility profiles with their snail hosts, and complete life cycles remain mysteries in many parts of the world. Surveys of digenean biology and ecology provide further insight and perspective into just how incredibly diverse and important helminth parasites are in shaping local ecosystems. Past surveys have provided substantial characterizations of adult digeneans within their definitive hosts, and many now have contributed toward furthering our understanding of larval digeneans within their intermediate host communities. However, much information about the diversity of digeneans and their relationships with their snail intermediate hosts are lacking in many locations across the globe. This is certainly true in Canada, where few records related to digenean-snail relationships exist. Currently, there is a need for more information about the presence and distribution of digeneans across Canada, and how this compares to other parts of North America and beyond. To address this diversity gap in Western Canada, six lakes within central Alberta were surveyed for the presence of snails and larval digenean species and their associations. This investigation into the diversity of digeneans utilized a combined approach of morphological and molecular tools to identify 39 digenean species among five snail host species, from biweekly collections, taken over the course of 2 years (2013–2014). Here, digenean-snail combinations, presence, and distribution across sampling sites and lakes are reported. Overall, this survey contributes new information toward digenean-snail compatibility, life cycles, and distribution in Northern lake ecosystems within North America.

Metalloproteases (MPs) have demonstrated roles in immune modulation. In some cases, these enzymes are produced by parasites to influence host immune responses such that parasite infection is facilitated. One of the best examples of parasite-mediated immune modulation is the matrix metalloprotease (MMP) leishmanolysin (Gp63), which is produced by species of the genus Leishmania to evade killing by host macrophages. Leishmanolysin-like proteins appear to be quite common in many invertebrates, however our understanding of the functions of these non-leishmania enzymes is limited. Numerous proteomic and transcriptomic screens of schistosomes, at all life cycle stages of the parasite, have identified leishmanolysin-like MPs as being present in abundance; with the highest levels being found during the intramolluscan larval stages and being produced by cercaria. This study aims to functionally characterize a Schistosoma mansoni variant of leishmanolysin that most resembles the enzyme produced by Leishmania, termed SmLeish. We demonstrate that SmLeish is an important component of S. mansoni excretory/secretory (ES) products and is produced by the sporocyst during infection. The presence of SmLeish interferes with the migration of Biomphalaria glabrata haemocytes, and causes them to present a phenotype that is less capable of sporocyst encapsulation. Knockdown of SmLeish in S. mansoni miracidia prior to exposure to susceptible B. glabrata reduces miracidia penetration success, causes a delay in reaching patent infection, and lowers cercaria output from infected snails., Author summary Parasitic flatworms, or digenetic trematodes, cause a wide range of diseases of both medical and agricultural importance. Nearly all species of digenea require specific species of snail for their larval development and transmission. The factors underpinning snail host specificity and how they dictate infection establishment and maintenance are interesting areas of research, both from the perspective of evolutionary immunology and potential application in the design of tools that aim to prevent trematode transmission. Currently, our understanding of snail-trematode associations is one-sided, being predominantly derived from studies that have focused on the snail immune response, with almost nothing known about how the parasite facilitates infection. Metalloproteases, such as leishmanolysin, are proteolytic enzymes; some of which are produced by parasites to influence host immune responses and facilitate parasite success upon encountering the host defense response. Here, we have functionally characterized a leishmanolysin-like metalloprotease (SmLeish) from Schistosoma mansoni, a causative agent of human schistosomiasis, which afflicts over 260 million people globally. We demonstrate that SmLeish is associated with developing sporocysts and is also located in S. mansoni excretory/secretory products and interferes with snail haemocyte morphology and migration. Knockdown of SmLeish in S. mansoni miracidia prior to exposure to Biomphalaria glabrata snails reduces miracidia penetration success, delays attainment of patent infections, and lowers cercaria output from infected snails.

Successful colonization of a compatible snail host by a digenetic trematode miracidium initiates a complex, proliferative development program requiring weeks to reach culmination in the form of production of cercariae which, once started, may persist for the remainder of the life span of the infected snail. How are such proliferative and invasive parasites able to circumvent host defenses and establish chronic infections? Using a microarray designed to monitor the internal defense and stress-related responses of the freshwater snail Biomphalaria glabrata, we have undertaken a time course study to monitor snail responses following exposure to two different trematode species to which the snail is susceptible: the medically important Schistosoma mansoni, exemplifying sporocyst production in its larval development, or Echinostoma paraensei, representing an emphasis on rediae production in its larval development. We sampled eight time points (0.5, 1, 2, 4, 8, 16 and 32 days p.i.) that cover the period required for cercariae to be produced. Following exposure to S. mansoni, there was a preponderance of up-regulated over down-regulated array features through 2 days p.i. but by 4 days p.i. and thereafter, this pattern was strongly reversed. For E. paraensei, there was a preponderance of down-regulated array features over up-regulated features at even 0.5 days p.i., a pattern that persists throughout the course of infection except for 1 day p.i., when up-regulated array features slightly outnumbered down-regulated features. Examination of particular array features revealed several that were up-regulated by both parasites early in the course of infection and one, fibrinogen related protein 4 (FREP 4), that remained significantly elevated throughout the course of infection with either parasite, effectively serving as a marker of infection. Many defense-related transcripts were persistently down-regulated, including several fibrinogen-containing lectins and homologs of molecules best known from vertebrate phagocytic cells. Our results are consistent with earlier studies suggesting that both parasites are able to interfere with host defense responses, including a tendency for E. paraensei to do so more rapidly and strongly than S. mansoni. They further suggest mechanisms for how trematodes are able to establish the chronic infections necessary for their continued success.

Mammalian macrophage CSF (MCSF; CSF-1) is the primary regulator of the mononuclear phagocyte lineage. We, for the first time, report the complete sequencing of five MCSF cDNAs from three fish species, rainbow trout, zebrafish, and goldfish. Despite the difference in the lengths of the MCSF transcripts, all of the fish MCSF molecules encode a signal peptide, a CSF-1 domain, a transmembrane domain, and an intracellular region. Each fish MCSF gene has a unique exon/intron structure. The primordial MCSF gene may have had a nine exon/eight intron structure. In this model, insertion of an intron in exon 6 in primitive fish created the fish type I MCSF, while the loss of this exon or part of the original exon 6 created the fish type II MCSF. Investigation of alternative splicing variants in trout suggests that no mammalian equivalent splice variants exist. The two trout MCSF genes are differentially expressed in vivo and contributed differently to the high-level expression of MCSF in spleen and head kidney. In contrast to the up-regulation of MCSF by PMA in mammals, in trout MCSF1 expression is down-regulated by PMA treatment. As in mammals, recombinant trout MCSF1 can promote the growth of head kidney leukocytes, and it up-regulates the expression of CXCR3 in head kidney macrophages, with the latter suggesting a role of MCSF in the trafficking of macrophages to sites of inflammation or injury where the CXCR3 ligands are expressed. Thus MCSF has an important role in the immune system of fish as in mammals.

Colony-stimulating factor-1 (CSF-1) regulates mononuclear cell proliferation, differentiation, and survival. The functions of CSF-1 are well documented in mammals; however, little is known about CSF-1 biology in lower vertebrates. This is the first report on the identification and functional characterization of a fish CSF-1 molecule expressed highly in the spleen and in phorbol 12-myristate 13-acetate-stimulated monocytes. Goldfish CSF-1 is a 199-amino acid protein that possesses the required cysteine residues to form important intra-chain and inter-chain disulfide bonds that allow CSF-1 to form a functional homodimer and to interact with its high affinity receptor, CSF-1R. Recombinant goldfish CSF-1 formed a homodimer and bound to the soluble goldfish CSF-1R. The addition of the recombinant CSF-1 to sorted goldfish progenitor cells, monocytes, and macrophages induced the differentiation of monocytes into macrophages and the proliferation of monocyte-like cells. The proliferation of these cells was abrogated by addition of an anti-CSF-1R antibody as well as the soluble CSF-1R. The ability of the soluble CSF-1R to inhibit CSF-1-induced proliferation represents a novel mechanism for the regulation of CSF-1 function.

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.

Over 100 years after the first description of macrophages by Metchnikoff, there are still questions as to the mechanisms leading to the heterogeneity of their lineage. Current views are based on the mononuclear phagocyte system (MPS) theory, where all mammalian macrophages are derived from circulating blood monocytes and ultimately from hematopoietic stem cells in the bone marrow. Our studies on the regulation of fish macrophage development, suggested that teleosts have alternate pathways of monopoiesis, which undoubtedly contribute to macrophage heterogeneity in the goldfish. Macrophage heterogeneity has been attributed to a network of positive and negative regulators of macrophage development, including soluble mediators known as colony-stimulating factors of which two (M-CSF and GM-CSF) promote formation and growth of mature macrophages. In contrast to our knowledge of CSFs and their receptors in mammals, there is no published information about fish macrophage CSFs. Since fish macrophages generate their own growth factors, it is reasonable to assume that pathways of fish macrophage development and hematopoiesis may be distinct from those of mammalian macrophages. More importantly, the presence of fish progenitor/stem cells and developing macrophages in long-term cultures, allowed us to address pathways of macrophage differentiation, which could not be addressed in mammalian macrophage culture systems. Characterization of primary kidney macrophage (PKM) cultures from goldfish hematopoietic tissues (kidney) indicated that three distinct subpopulations developed in response to endogenous macrophage growth factors. These macrophage subpopulations expressed several differentiation markers, including the hematopoietic stem cell antigen AC133, c-kit, granulin, CD63, macrosialin, c/EBPbeta, legumain, and the colony-stimulating factor receptor-1 (CSF-1R). In the goldfish, there appeared to be a stringent control between those early progenitors that self-renewed, and those that were recruited into the maturation pathways. We report that upon commitment, goldfish macrophages developed through two distinct differentiation pathways: one consistent with the "classical" pathway (MPS) of macrophage development (progenitors-->monocytes-->mature macrophages), and an "alternate" pathway (AP-macrophages) where mature macrophages appeared to rapidly develop from early progenitors in the absence of an intermediate monocyte stage. AP-macrophages represent a unique subset of spontaneously growing cells. Their self-renewal was promoted by endogenous macrophage growth factors (MGF), and effectively controlled by a novel soluble form of the CSF-1R (sCSF-1R). The discovery of sCSF-1R in the goldfish highlights the inherent complexity in the hematopoietic regulatory machinery of teleosts.

Nature Communications 8: Article number: 15451 (2017); Published 16 May 2017; Updated 23 August 2017 The original version of this Article contained an error in the spelling of the author Leon Di Stefano, which was incorrectly given as Leon di Stephano. This has now been corrected in both the PDF andHTML versions of the Article.

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished., Author Summary Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Understanding the factors important for snail resistance to schistosome infection will facilitate new lines of investigation to 1) understand the underlying basis of compatibility between schistosomes and snails in endemic areas and how this affects transmission dynamics and control efforts; and 2) to reveal ways to manipulate natural snail populations to enhance their resistance to schistosome infections. Here, we present the first evidence that a snail immune molecule, fibrinogen related protein 3 (FREP3), is important for successful defense against schistosome infections in Biomphalaria snails. In addition, we demonstrate that FREP3 is a target suppressed by trematode parasites to facilitate their establishment within the snail.

The Clark P. Read award is given to an individual who has demonstrated extraordinary leadership in the training of young scientists. In addition, the individual must have influenced the research and graduate education of a department to increase the number of students completing graduate training in disciplines of parasitology. It is an honor to be here before you all today to introduce the recipient of this year’s Clark P. Read Mentor Award, Sam Loker, from the University of New Mexico. Sam has been an outstanding mentor for me not only during my time in his lab but also currently as I begin my journey mentoring my own students. I find that I often ponder how Sam would handle particular situations; for example, motivating students, tailoring training to their personalities, and engaging students in areas outside of pure scientific curiosity. I cannot think of anyone more deserving of this recognition. As I have begun to embark upon my own journey of mentoring students and trainees, I have come to appreciate how challenging the process of mentorship can be. I have come to realize that I have been incredibly lucky over my academic career, having been exposed to incredible mentorship that has set a high bar for me to strive to achieve. Mentors like Sam make the mentorship process look natural. They possess intrinsic qualities that allow them to understand people, and more importantly, make others know that they are valued and being understood. Of course, mentorship does not simply relate to the ability of a mentor to properly train a mentee in the technical aspects of science. An excellent mentor, one worthy of the Clark P. Read Award, is one that does not simply mentor toward academic scientific careers, but provides mentorship tailored to the individual by nurturing scientific curiosity as well as personal development. Sam is able to draw from his experiences to provide insight on many aspects of life, and often I found Sam serving as a role model as well as a scientific mentor. His ability to demonstrate empathy and relate to his mentees makes Sam an easy individual to listen to and to trust. Empirically, Sam has achieved an incredible level of mentorship success. Sam has advised 12 Ph.D. students, 12 M.S. students, 15 postdoctoral fellows, and he has published more than 147 peerreviewed manuscripts with one or more of those trainees. In addition to this list are the many international students and researchers that Sam has mentored, particularly a large group of researchers from Kenya, where Sam has undertaken many schistosome-related projects. One of these students, Eric Lelo, wrote a letter in support of Sam’s nomination and emphasized in the letter that Sam played an essential role in his training. Also worth noting are the countless undergraduate students that Sam has brought through his lab. These students are often exposed to parasitology research before ever considering graduate school; and in some cases, their experience in Sam’s lab encouraged them to pursue graduate research. As a secondary metric of mentoring success, Sam has mentored more than 14 scientists that now maintain an academic researchfocused appointment, and almost all of these individuals continue to work in parasitology. An additional 5 hold lecturer positions and are undoubtedly encouraging the development of the next generations of scientists. It is important to note that Sam’s mentorship extends well beyond those individuals that train directly within his lab. Two particularly noteworthy undertakings that Sam has spearheaded have and will continue to have a lasting impact on parasitological research at the University of New Mexico. First, Sam is primarily responsible for the creation and maintenance of an incredibly successful collaborative research center at UNM that has recruited exceptional trainees and faculty to UNM under the banner of the Center for Evolutionary and Comparative Immunology (CETI). CETI is funded by the NIH Centers for Biomedical Research Excellence (COBRE) funding scheme and provides support for trainees in several ways, as well as seed grants for young investigators and state-of-the-art core facilities that can be used by CETI and UNM researchers and trainees. Sam plays a central mentorship role to many students and young faculty that have been recruited to CETI, and CETI has provided a fruitful training environment, through which many of Sam’s trainees have passed. Second, Sam has played a pivotal role in establishing the Parasite Collection at the Museum of Southwest Biology. This collection has served as a parasite resource for numerous researchers, and it will continue to support parasitological research for years to come. In summary, I believe that Sam embodies all of the characteristics of an outstanding mentor. A common theme found in all letters supporting Sam’s nomination for this award focused on his willingness to commit a seemingly endless amount of time to his mentees, support balance in their lives, and display a genuine interest in their activities outside of the lab. His commitment to treating mentees with respect, and as peers, has led to many lasting friendships that can be traced back to Sam’s mentorship approach. Sam’s trainees have come from many countries and cultures. Although many have had the benefit of learning from Sam at UNM, many have also learned under his tutelage in Kenya, Tanzania, Nepal, or elsewhere. It is perhaps this flexibility, Sam’s capacity to connect and understand all sorts of individuals, from all over the world that underscores his talent as a mentor. Please join me in congratulating Sam, the 2015 recipient of the Clark P. Read mentorship award.