You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction {#s1}
============

Glycans are complex carbohydrate (CHO) chains normally covalently bound to polypeptides, lipids or other carrier molecules. Glycoconjugates such as glycoproteins, glycolipids and proteoglycans represent one of the most prominent classes of molecules exhibited by schistosomes. Schistosome glycans are highly diverse structurally and have been implicated in a variety of physiological processes during schistosome infection of its mammalian host, most notably their involvement in modulating protective immune responses and immunopathology (see reviews [@pntd.0001569-Hokke1]--[@pntd.0001569-vanDie1]). Similarly glycans are also highly expressed in the free-swimming miracidial and intramolluscan developmental stages of *Schistosoma* spp. as shown by earlier exogenous lectin-binding studies [@pntd.0001569-Yoshino1], [@pntd.0001569-Uchikawa1], and more recent glycotope/glycomic analyses [@pntd.0001569-Nyame1]--[@pntd.0001569-Peterson1]. However, despite the presence of diverse glycans associated with the larval surface and its secretions/excretions, their functional significance remains unknown. A popular notion that recently has gained traction in the *Biomphalaria glabrata-Schistosoma mansoni* system poses that larval glycans and/or their associated glycoconjugates may be serving as pathogen-associated molecular patterns (PAMPs) that interact with lectin-like pathogen recognition receptors (PRRs), thereby mediating innate immune responses to invading miracidia (see reviews [@pntd.0001569-Adema1]--[@pntd.0001569-Yoshino2]). This concept has been incorporated into a proposed mechanism, termed compatibility polymorphism [@pntd.0001569-Mitta1], in which it is hypothesized that high molecular diversity in relevant PAMP and PRR systems can provide the necessary variation in receptor-ligand interactions to account for differences in infection rates seen in different snail-schistosome strain combinations [@pntd.0001569-Theron1]. Two candidate gene families that fulfill the basic requirements of exhibiting high molecular polymorphism and potential functional diversity are the fibrinogen-related proteins or Freps, lectin-like proteins in plasma of *B. glabrata* snails [@pntd.0001569-Adema2] and a family of polymorphic mucins from *S. mansoni* (*Sm*PoMuc; [@pntd.0001569-Roger1]). Recent studies have reported the selective ability of *Sm*PoMuc to form complexes with Freps from snail plasma [@pntd.0001569-Mone1], as well as the demonstration of a direct linkage between expression of one *B. glabrata* Frep (Frep 3) and resistance to trematode infection [@pntd.0001569-Hanington1], thus supporting a functional basis for the compatibility polymorphism hypothesis. The specific ligands mediating Frep-*Sm*PoMuc binding, however, still remain unknown, although the lectin-like properties of Freps and the fact that *Sm*PoMuc are highly glycosylated point to the likelihood that specific glycan structures may be serving to mediate larval recognition, leading to hemocytic encapsulation and parasite killing typical of resistant host phenotypes [@pntd.0001569-Bayne2], [@pntd.0001569-Boswell1]. Taken further it may then be predicted that the absence of relevant snail Freps and/or differences in expressed glycans (e.g., based on variable *Sm*PoMuc expression) should lead to the opposite schistosome-snail outcome; that is, larval survival due to nonrecognition by the snail\'s immune system. Assuming snail-schistosome compatibility involves a diversified lectin-based immunorecognition system, the repertoire of larval-expressed glycans (both qualitative and quantitative) potentially has a direct impact on hemocyte reactivity towards a given parasite within the snail host. Following up on Damian\'s "molecular mimicry" hypothesis [@pntd.0001569-Damian1], [@pntd.0001569-Damian2], that a parasite constitutively expressing host-like molecules could render the host immunologically "blind" to the parasite\'s presence, it has been suggested that early invading larvae (miracidia, sporocysts) possessing shared antigenic structures with their host snail may evade recognition by the internal defense system [@pntd.0001569-Yoshino3], [@pntd.0001569-Bayne3]. Although earlier studies demonstrated serological reactivity between larval *S. mansoni* and snail host hemolymph [@pntd.0001569-Yoshino4], [@pntd.0001569-Bayne4], Dissous et al. [@pntd.0001569-Dissous1] were the first to show that shared CHOs are represented among those immunoreactive epitopes. Recent structural analyses of N-glycans from *B. glabrata* plasma (cell-free hemolymph) provide definitive evidence that glycan structures, specifically terminal fucosylated LacdiNAc variants and core-linked xylose are shared between *S. mansoni* and its snail host [@pntd.0001569-Lehr1]. In follow-up studies using highly specific monoclonal antibodies (mABs) to these, and other CHO epitopes (glycotopes), extensive crossreactivity has now been confirmed between larval glycans and those of various *B. glabrata* tissues [@pntd.0001569-Lehr1], [@pntd.0001569-Peterson1], [@pntd.0001569-Lehr3], notably between host hemolymph and proteins released during larval transformation [@pntd.0001569-Peterson1]. During the first hours following miracidial entry into the snail host, a complex molecular interplay takes place in which an array of macromolecules are released during miracidium-to-sporocyst transformation [@pntd.0001569-Guillou1], [@pntd.0001569-Wu1]. As a consequence, newly developing primary sporocysts are enveloped in a glycan-rich localized environment comprised mainly of glycoproteins, but also may comprise other glycoconjugates. These larval transformation products or LTPs [@pntd.0001569-Wu1], in addition to serving as a passive source of host mimicked" molecules, also may actively bind snail lectins (e.g., Freps; [@pntd.0001569-Adema2]), thereby blocking lectin reactivity against newly developing sporocysts [@pntd.0001569-Roger2]. Given the possible immune modulating effects of LTPs released at a critical time when schistosome miracidia/sporocysts are in the process of establishing infections in the snail host, the present study investigated the effect of LTP exposure on the profile of shared glycotopes associated with plasma from susceptible (NMRI) and resistant (BS-90) strains of *B. glabrata*. Results of these experiments demonstrate that glycoconjugates released during *S. mansoni* larval transformation significantly alter patterns of shared plasma protein glycotopes by either binding and blocking, or by exposing them, thereby providing a possible mechanism by which molecules released by early developing larvae may impact initial immune interactions at the host-parasite interface.

Materials and Methods {#s2}
=====================

Ethics statement {#s2a}
----------------

All experimental protocols involving mice and rabbits used in the course of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison under Animal Welfare Assurance No. A3368-01.

Preparation of snail plasma {#s2b}
---------------------------

Hemolymph was obtained from *S. mansoni*-susceptible and -resistant *B. glabrata* snails (NMRI and BS90 strains, respectively) by the headfoot retraction method [@pntd.0001569-Sminia1]. Hemolymph from approximately 150 snails (12--15 mm shell diameter) of each strain were pooled and represented a common source for subsequent Western and far-Western blot analyses. During the course of this study a second replicate pool of hemolymph was obtained from which immunoblot analyses were repeated. Upon collection, hemolymph of each strain was dispensed into 1.5 mL microcentrifuge tubes containing cold, sterile Chernin\'s balanced salt solution (CBSS; [@pntd.0001569-Chernin1]) creating a 1∶1 dilution of CBSS:hemolymph. Tubes were centrifuged at 260× g (Eppendorf 5810R, Hauppauge, NY) for 10 min to pellet hemocytes followed by transfer of the cell-free hemolymph (plasma) to a 15-mL Amicon centrifugal ultrafiltration tube (Amicon Ultra-100 k; Millipore Corp, Billerica, MA) with a nominal molecular weight cut-off of 100 kDa. Tubes were centrifuged at 1600× g for 95 min at 4°C (Eppendorf 5810R). This plasma ultrafiltration step was performed in order to separate the snail hemoglobin (which comprises the predominant protein constituent of hemolymph) from a hemoglobin-depleted lower molecular weight plasma fraction. Fractions were designated "\>100 kDa-fraction", which contained the vast majority of hemoglobin as indicated by its intense red color, and "\<100 kDa-fraction", which typically was colorless. Concentration of the \<100 kDa-fraction was then carried out using a 3 kDa MW cutoff centrifugal ultrafiltration tube (Amicon Ultra-3 k; Millipore Corp, Billerica, MA). Protein concentrations were determined using a Nanodrop spectrophotometer (ND-1000; Nanodrop Technologies, Wilmington, DE), followed by addition of protease inhibitors (Protease Inhibitor Cocktail SetIII, EDTA-free; Calbiochem, San Diego, CA) to protect proteins from endogenous protease activities.

Preparation of *S. mansoni* larval transformation products (LTPs) {#s2c}
-----------------------------------------------------------------

Miracidia were obtained from eggs recovered from infected mice provided by the Biomedical Research Institute (BRI, Rockville, MD) and housed at the University of Wisconsin Charmany Instructional Facility. Miracidia were isolated under axenic conditions as previously described [@pntd.0001569-Yoshino5] and placed into *in vitro* culture in CBSS supplemented with penicillin and streptomycin and 1 g/L each of glucose and trehalose (CBSS+) under normoxic conditions at 26°C. Culture supernatants containing larval transformation products (LTPs) were harvested after 24 hr by filtering through a Whatman Puradisc 0.2-µm syringe filter (GE Healthcare LTD, Buckinghamshire, UK) to remove stray sporocysts, epidermal plates, and any cellular debris. Culture supernatants were then concentrated by ultrafiltration (Amicon Ultra-3 k; Millipore Corp, Billerica, MA) and protein concentration determined using a Nanodrop ND-1000. Following addition of protease inhibitors as described above, aliquots were stored at 4°C if used within a week, or at −80°C for longer storage periods. Before use in far-Western blot analyses, concentrated LTP was diluted in snail Tris-buffered saline (sTBS; 20 mM Tris-HCl, 150 mM NaCl, pH7.4).

Western and far-Western blot analyses {#s2d}
-------------------------------------

### Anti-LTP polyclonal and anti-glycan monoclonal antibodies {#s2d1}

Production of the polyclonal anti-LTP antiserum is detailed in Wu et al. [@pntd.0001569-Wu1]. Briefly, acetone-precipitated LTP (250 µg in Freund\'s complete adjuvant) was injected subcutaneously into multiple sites of New Zealand white rabbits, followed by two booster injections using Freund\'s incomplete adjuvant (Panigen, Blanchardville, WI). Antiserum, harvested from the highest titered rabbit, was applied to a Protein G/Protein A-agarose column (Calbiochem, La Jolla, CA) to isolate the IgG fraction, which was then aliquoted and stored in 0.1% azide at 4°C. IgG isolated from pre-immune serum was used as a negative control.

Monoclonal antibodies (mABs) specifically reactive to a variety of terminal di-, tri- and tetrasaccharide elements of schistosome glycans (listed in [Table 1](#pntd-0001569-t001){ref-type="table"}) were produced and characterized as previously described [@pntd.0001569-Robijn1]--[@pntd.0001569-vanRoon1].

10.1371/journal.pntd.0001569.t001

###### Glycotope specificities of anti-glycan monoclonal antibodies.

![](pntd.0001569.t001){#pntd-0001569-t001-1}

  MAB Number             Ig Class                         Glycotope Structure                               Glycotope Name
  --------------------- ---------- ------------------------------------------------------------------ ---------------------------
  259-2A1/273-3F2        IgG3/IgM                         GalNAc(β1--4)GlcNAc                               LacdiNAc (LDN)
  114-4E8-A                IgM                     GalNAc(β1--4)\[Fuc(α1--3)\]GlcNAc                             LDN-F
  258-3E3                  IgM                     Fuc\[(α1--3)\]GalNAc(β1--4)GlcNAc                             F-LDN
  128-1E7-C                IgM                Fuc(α1--3)GalNAc(β1--4)\[Fuc(α1--3)\]GlcNAc                       F-LDN-F
  114-5B1-A/290-4A8      IgG1/IgM             GalNAc(β1--4)\[Fuc(α1--2)Fuc(α1--3)\]GlcNAc                       LDN-DF
  114-4D12-A               IgG3     Fuc(α1--2)Fuc(α1--3)GalNAc(β1--4) \[Fuc(α1--2)Fuc(α1--3)\]GlcNAc           DF-LDN-DF
  100-4G11-A               IgM           Man(α1--6)\[Man α1--3)\]Man(β1--4) GlcNAc(β1--4)GlcNAc        Trimannosyl core (TriMan)
  128-4F9-A/291-2G3-A    IgM/IgG1                     Galβ1--4\[Fuc(α1--3)\]GlcNAc                           Lewis X (LeX)

Schistosome glycan-specific monoclonal antibodies (MABs) were used in Western and far-Western blot analyses to demonstrate the occurrence of shared glycans on plasma proteins from the snail *Biomphalaria glabrata*. MABs were generated from *Schistosoma*-infected mice and their specificities characterized [@pntd.0001569-Robijn1]--[@pntd.0001569-vanRoon1] by screening a panel of synthetic neoglycoproteins bearing schistosome-related glycan determinants.

### Western/far-Western blot analyses {#s2d2}

As a preliminary step to investigating specific glycotope sharing between *B. glabrata* plasma and larval schistosomes, equal concentrations (15 µg in 18 µL/well) of BS-90 and NMRI plasma fractions were separated by SDS-PAGE and subjected to Western blot analysis using the polyclonal anti-LTP antiserum [@pntd.0001569-Wu1]. Anti-LTP Western blots were performed to show that a broad range of LTP epitopes were shared with *B. glabrata* plasma and these epitopes were similarly distributed between plasma proteins of both snail strains. In addition, to compare the protein content and general protein profiles exhibited by BS-90 and NMRI plasma samples, SDS-PAGE separated plasma from both snail strains were stained with Coomassie brilliant blue and silver stains. These initial protein analyses were done to demonstrate that experiments were being performed on NMRI and BS-90 plasma pools that exhibited similar protein banding profiles.

In order to determine the distribution of specific schistosome glycotopes naturally occurring among *B. glabrata* plasma proteins the following protocol was employed: Plasma samples from each fraction (\> and \<100 kDa fractions) were loaded onto separate 15-well 12.5% SDS-PAGE slab gels with samples from the two snail strains in alternating lanes. This permitted side-by-side qualitative and semi-quantitative comparisons of NMRI and BS-90 plasma reaction profiles for each antibody. After separation and blotting, nitrocellulose membranes were cut into strips, each containing paired NMRI/BS-90 plasma lanes and MW markers, followed by incubation in blocking buffer (5% BSA in sTBS, pH7.4) overnight at 4°C. Blots were then washed 5× in sTBST (sTBS/0.05%Tween 20), incubated in anti-glycan mAB (1∶100 dilution of 5× concentrated mAB culture supernatants) for 2 hr at 22°C, followed by an alkaline phosphatase (AP)-conjugated rabbit anti-mouse Ig antiserum and finally treated with AP color developing reagents (Pierce, Rockford, IL) to visualize immunoreactivities. Due to the absence of LeX antibody reactivity to native plasma proteins from both snail strains, this mAB served as a negative antibody control.

In conjunction with the above direct assessment of shared larval glycotopes on plasma proteins, the effects of LTP exposure on the profiles of shared plasma glycotopes was determined using a far-Western blot approach [@pntd.0001569-Edmondson1]. Far-Western blot methods are commonly used to test for protein-protein interactions, although interactions between proteins and other molecules may also be assessed. In our far-Western blot assays interactions between plasma proteins and LTPs were assessed. SDS-PAGE-separated and blotted paired NMRI and BS-90 plasma samples were incubated in blocking buffer overnight, washed, and exposed to LTP (50 µg/mL blocking buffer) overnight at 4°C. This was followed by 5 washes in sTBST and 2 hr incubation in anti-glycan mABs at 22°C. Blots were then processed for glycotope reactivity as described above. Western and far-Western blot analyses (using paired plasma samples) for each of the mABs were repeated 2--3 times with similar results. An assumption is made in these assays that upon transfer of plasma protein to nitrocellulose membranes and removal of SDS, polypeptides will reconfigure to their native conformation. Although we realize that reconfiguration may not occur for all proteins, especially those with disulfide-dependent multimeric structures, it is believed that this approach still provides an accurate picture of putative LTP-plasma binding interactions using a comparative experimental design.

Results {#s3}
=======

Subjecting *B. glabrata* plasma to centrifugal molecular filtration using 100 kDa MW spin-filters generally was effective in separating intact plasma into fractions enriched for proteins above ∼75 kDa (\>100 kDa cut-off) and those of 50 kDa and lower ([Figure 1A](#pntd-0001569-g001){ref-type="fig"}). Because these ultrafiltration membranes only provide a nominal MW cut-off, clearly there is considerable MW overlap between the high and low MW fractions. The overlap of mid-range MW proteins in both fractions (25--75 kDa) also may have been the result of breakdown of high MW complexes in the \>100 kDa fraction during SDS-PAGE processing. Importantly, however, based on protein banding patterns revealed in Coomassie blue- and silver-stained SDS-PAGE gels, only minor differences in protein molecular mass profiles were evident between NMRI and BS-90 snail strains. When separated and blotted snail plasma was probed with a polyclonal antiserum generated against products released during *in vitro* miracidial transformation (LTP), there was extensive immunoreactivity with plasma in both high (\>100 kDa) and low (\<100 kDa) molecular weight plasma fractions ([Figure 1B](#pntd-0001569-g001){ref-type="fig"}). Reactivity of our anti-LTP polyclonal antiserum to blotted plasma provided a confirmation that plasma proteins did in fact share crossreactive antigens (epitopes) with products released by *S. mansoni* larvae during early larval development. Because few differences in crossreactive bands were observed between NMRI and BS-90 plasmas using the polyclonal antibody to undefined antigens contained in the LTP, we sought to incorporate highly specific mAB to defined epitopes to identify possible snail strain differences in shared determinants. The similarity in crossreactive plasma profiles also served as an additional sample loading control, allowing for comparisons of mAB reactivities between snail strain samples. These initial analyses of snail plasmas established that (1) NMRI and BS-90 plasma have similar protein content (loading control), (2) that plasma and LTPs did, in fact, share an extensive array of undefined epitopes, and (3) that the use of specific mABs to defined epitopes could provide for a detailed comparison of the shared epitopes exhibited by NMRI and BS-90 snail plasmas in the presence and absence of LTP.

![Initial comparison of plasma protein samples from *Biomphalaria glabrata* snail strains.\
Plasma (cell-free hemolymph) from the NMRI (N) and BS-90 (B) strains of *B. glabrata* was separated into \<100 kDa and \>100 kDa fractions by molecular ultrafiltration, followed by separation by SDS-PAGE and staining with Coomassie brilliant blue or silver ([Figure 1A](#pntd-0001569-g001){ref-type="fig"}). [Figure 1B](#pntd-0001569-g001){ref-type="fig"} is a Western blot of NMRI and BS-90 plasma fractions (\</\>100 kDa) probed with a polyclonal antiserum to *Schistosoma mansoni* larval transformation products to confirm that epitope sharing in fact occurs between plasma and larval products. Note that plasma from NMRI and BS-90 snails exhibit similar patterns of protein banding (A) and anti-larval immunoreactivity (B) indicating comparable sample loading and the presence of shared epitopes. Molecular weight markers are indicated on the right.](pntd.0001569.g001){#pntd-0001569-g001}

In contrast to polyclonal anti-LTP staining, the immunoreactivities of 8 glycan-specific mABs with native (untreated) *B. glabrata* plasma proteins were highly varied as shown by Western blot analyses. Highest reactivity, both in staining intensity and number of proteins, was observed using the trimannosyl N-glycan core (TriMan) mAB, while no staining was seen with anti-Lewis X (LeX) ([Figure 2](#pntd-0001569-g002){ref-type="fig"}; −LTP). Snail strain differences in staining patterns for TriMan were detected including greater reactivity with NMRI proteins between 50--75 kDa ([Figure 2](#pntd-0001569-g002){ref-type="fig"}; −LTP/\<100 kDa fraction) and in BS-90, proteins between 15--20 kDa ([Figure 2](#pntd-0001569-g002){ref-type="fig"}; −LTP/\<100 kDa fraction). Moreover, pre-incubation of the blotted plasma proteins with *S. mansoni* LTP had little effect on the patterns of shared TriMan glycotopes, with the exception of enhanced staining of BS-90 proteins in the 25--37 kDa range for TriMan ([Figure 2](#pntd-0001569-g002){ref-type="fig"}; +LTP/\>100 kDa fraction). Similarly, interactions of LTP with high MW BS-90 plasma proteins resulted in the appearance of anti-LeX-reactive bands between 50--150 kDa ([Figure 2](#pntd-0001569-g002){ref-type="fig"}; +LTP/\>100 kDa fraction).

![Western/far-Western blot analyses of Lewis X and trimannosyl core glycotopes on plasma proteins.\
Fractionated *Biomphalaria glabrata* plasma (\>100 kDa; \<100 kDa) obtained from the NMRI (N) and BS-90 (B) snail strains were separated, blotted to nitrocellulose and pre-incubated in the presence (+LTP) or absence (−LTP) of *S. mansoni* larval transformation products (LTP). LTP-exposed (far-Western) and unexposed (Western) blots were probed with specific anti-glycan mABs against the N-linked trimannosyl core glycan (TriMan) and the Lewis X antigen (see [Table 1](#pntd-0001569-t001){ref-type="table"} for glycan structures). Molecular weight markers are indicated on the right.](pntd.0001569.g002){#pntd-0001569-g002}

Similar to TriMan mAB reactivity, consistent qualitative and/or quantitative differences in shared glycotope distribution between native (untreated) NMRI and BS-90 plasma proteins were observed for LDN, F-LDN and F-LDN-F glycotopes, especially in the \<100 kDa fraction ([Figure 3](#pntd-0001569-g003){ref-type="fig"}; −LTP/\<100 kDa fraction). Typically, NMRI exhibited higher levels and/or wider distribution of these glycotopes when compared to BS-90 proteins, and notably displayed unique patterns of glycotope reactivity on native proteins indicating that shared glycotopes on NMRI proteins were either associated with unique subsets of glycoproteins or differed quantitatively in their occurrence on individual plasma proteins. Although the occurrence of LDN, F-LDN, LDN-F and F-LDN-F glycotopes varied considerably on plasma proteins contained in the \>100 kDa fraction ([Figure 3](#pntd-0001569-g003){ref-type="fig"}; −LTP/\>100 kDa), in the absence of LTP treatment, no consistent snail strain differences were observed for these higher MW proteins. Monoclonal antibodies to LDN-DF and DF-LDN-DF exhibited little reactivity against native plasma proteins, with the exception of higher MW polypeptides in the \>100 kDa fraction that stained with anti-DF-LDN-DF ([Figure 4](#pntd-0001569-g004){ref-type="fig"}; −LTP/\>100 kDa). Also, no notable snail strain differences in the distribution of these glycotopes were observed.

![Western/far-Western blot analyses of LDN and (Fucα1,3)-linked LDN glycotopes on plasma proteins.\
Fractionated *Biomphalaria glabrata* plasma (\>100 kDa; \<100 kDa) from the NMRI (N) and BS-90 (B) snail strains were probed with anti-glycan mABs recognizing LacdiNAc (LDN) and various (Fucα1,3)-linked forms of LDN (F-LDN, F-LDN-F and LDN-F; see [Table 1](#pntd-0001569-t001){ref-type="table"} for glycan structures and mABs used). Blots containing separated plasma proteins were pre-incubated in the presence (+LTP) or absence (−LTP) of *S. mansoni* larval transformation products (LTP) to determine the effects of LTP exposure on the distribution and intensity of shared glycotopes among proteins of the two *B. glabrata* strains. Molecular weight markers are indicated on the right.](pntd.0001569.g003){#pntd-0001569-g003}

![Western/far-Western blot analyses of (Fucα1,2/Fucα1,3)-linked LDN glycotopes on plasma proteins.\
Fractionated NMRI (N) and BS-90 (B) *Biomphalaria glabrata* plasma (\>100 kDa; \<100 kDa) were probed with schistosome glycan-specific mABs recognizing LacdiNAc (LDN) containing one or two α1,2-linked fucosyl-dimers (LDN-DF; DF-LDN-DF; see [Table 1](#pntd-0001569-t001){ref-type="table"} for structures and mAB used). Blotted plasma proteins were incubated in the presence (+LTP) or absence (−LTP) of *S. mansoni* larval transformation products (LTP) prior to mAB treatment to determine the effects of LTP exposure on the distribution and intensity of these glycotopes among plasma proteins. Molecular weight markers are indicated on the right.](pntd.0001569.g004){#pntd-0001569-g004}

Exposure of SDS-PAGE-separated and blotted snail plasma proteins to LTP had a profound effect on the reactivity of plasma proteins to the panel of anti-glycan mABs. Alterations in the exposed glycotope distribution on plasma proteins were manifested in several ways: i) decreased glycotope-specific antibody reactivity in selected plasma proteins as noted with F-LDN, F-LDN-F and DF-LDN-DF ([Figures 3](#pntd-0001569-g003){ref-type="fig"} and [4](#pntd-0001569-g004){ref-type="fig"}; +LTP/\>100 kDa fraction), ii) qualitative changes in antibody reactivity to plasma proteins when compared to non-LTP exposed blots, e.g., F-LDN and F-LDN-F ([Figure 3](#pntd-0001569-g003){ref-type="fig"}; +LTP/\<100 kDa fraction) or iii) appearance of specific glycotopes in previously undetected or existing protein bands. These include the reactivities of anti-LDN-F, LDN-DF and DF-LDN-DF in LTP-exposed plasma proteins in the \<100 kDa fraction ([Figs. 3](#pntd-0001569-g003){ref-type="fig"} and [4](#pntd-0001569-g004){ref-type="fig"}; +LTP/\<100 kDa) and anti-LeX, LDN, LDN-F and LDN-DF mAB reactions to the \>100 kDa fraction ([Figures 2](#pntd-0001569-g002){ref-type="fig"}, [3](#pntd-0001569-g003){ref-type="fig"} and [4](#pntd-0001569-g004){ref-type="fig"}; +LTP/\>100 kDa). Furthermore, it was noted that exposure of blotted NMRI and BS-90 plasma to LTP altered the native (non-treated) glycotope staining profiles for LDN-F, F-LDN, F-LDN-F, and LDN-DF ([Figures 3](#pntd-0001569-g003){ref-type="fig"} and [4](#pntd-0001569-g004){ref-type="fig"}; +LTP/\<100 kDa) and LeX ([Figure 2](#pntd-0001569-g002){ref-type="fig"}; +LTP/\>100 kDa), in a snail strain-specific manner. Examples of plasma proteins that exhibit snail strain-specific changes in glycotope immunoreactivity following LTP-exposure are listed in [Table 2](#pntd-0001569-t002){ref-type="table"}.

10.1371/journal.pntd.0001569.t002

###### Differential effect of LTP-binding on plasma glycotope immunoreactivity.

![](pntd.0001569.t002){#pntd-0001569-t002-2}

  Glycotope    NMRI *B. glabrata*   BS-90 *B. glabrata*               
  ----------- -------------------- --------------------- ------------ -------------
  LeX                  \-                   \-                \-       55, 80, 150
  TriMan               \-                   \-                \-       30, 32, 90
  F-LDN                37                   \-                \-           130
  LDN-F           25, 37, 100               \-                \-           \-
  F-LDN-F              \-                   \-               130           130
  LDN-DF               45                   \-            16, 18, 23       \-
  DF-LDN-DF            37                   \-                \-           \-

Listed are the estimated molecular weights (in kDa) of plasma proteins that exhibit snail strain-specific changes in glycotope immunoreactivity following exposure to larval transformation products (LTP). Table headings "\< or \>100 kDa" refer to the plasma fractions illustrated in [Figures 2](#pntd-0001569-g002){ref-type="fig"}, [3](#pntd-0001569-g003){ref-type="fig"}, [4](#pntd-0001569-g004){ref-type="fig"}, while "Glycotope" indicates the glycan epitope recognized by specific mABs (see [Table 1](#pntd-0001569-t001){ref-type="table"} for glycotope structure). Numbers represent approximate MWs of glycotope-bearing plasma proteins that differ between from NMRI and BS-90 *B. glabrata* snail strains.

Discussion {#s4}
==========

As alluded to in the introduction, the importance of glycans as potential PAMPs in this model, and probably other trematode-snail systems, has been highlighted in recent studies focusing on the identification of specific glycan structures associated with early developing larval stages of *S. mansoni* [@pntd.0001569-Nyame1], [@pntd.0001569-Lehr2], [@pntd.0001569-Peterson1], [@pntd.0001569-Lehr3]. In the present study, mABs generated from *Schistosoma*-infected mice and reactive for terminal elements of schistosome glycans expressed by miracidial and primary sporocyst stages [@pntd.0001569-Nyame1], [@pntd.0001569-Lehr2], [@pntd.0001569-Peterson1], exhibited extensive reactivity with cell-free hemolymph (plasma) glycoproteins of *B. glabrata*. In addition, based on the plasma protein binding patterns for selected glycotope-specific mABs, clear differences were observed between the NMRI and BS-90 snail strains; most notably NMRI plasma possessed a greater number and/or higher staining intensity of glycoproteins displaying the LDN, F-LDN and F-LDN-F glycotopes than plasma proteins from BS-90 snails. These findings are generally consistent with those reported by Lehr et al. [@pntd.0001569-Lehr3], although in the present study no consistent snail-strain differences were observed for shared LDN-F, LDN-DF, DF-LDN-DF on native plasma proteins.

Based on the results of these shared glycotope studies, a main goal of the present work was to determine if molecules released during early *S. mansoni* larval development (miracidium-to-sporocyst transformation) are capable of interacting with plasma proteins, and if so, what effect this interaction may have on the patterns of shared plasma glycans in these *B. glabrata* strains. The initial 24 hours of schistosome larval development is believed to be a critical period in determining the success or failure of establishing infections within the snail host [@pntd.0001569-Bayne1]. Therefore, unraveling the complex molecular interactions occurring during this time should provide important insights into the underlying mechanisms of host-parasite compatibility in this model system. Upon penetration of a susceptible snail host, miracidia rapidly (within hours) begin transforming to sporocysts by shedding their ciliated epidermal plates during the process of sporocyst tegument formation [@pntd.0001569-Pan1], [@pntd.0001569-Loker1]. As larvae transform they release a complex mixture of molecules (LTPs), including many glycoproteins [@pntd.0001569-Guillou1], [@pntd.0001569-Wu1], [@pntd.0001569-Lodes1], which are capable of binding snail hemolymph components [@pntd.0001569-Davids1], [@pntd.0001569-Johnson1] and modulating hemocyte function [@pntd.0001569-Yoshino2], [@pntd.0001569-Walker1]--[@pntd.0001569-Zahoor2], likely through the action of mimicked CHOs [@pntd.0001569-Plows1].

Since innate resistance to incompatible *S. mansoni* strains is mediated by hemocytic encapsulation, the molecular composition of LTPs confronting immune elements in hemolymph would be predicted to have functional consequences resulting from LTP binding to plasma or hemocyte proteins, thereby interfering with larval recognition mechanisms, or by altering hemolymph glycoprotein structures through enzymatic means. In the present study, we have shown that exposure of plasma proteins to LTP dramatically alters the patterns of anti-glycan mAB reactivity for selected glycotopes naturally-occurring (shared) on snail plasma proteins. The most notable effects of LTP exposure on shared glycotope immunoreactivity include reduced or enhanced immunostaining of existing reactive protein bands (e.g., LDN, F-LDN), and changes in existing patterns of plasma glycotope reactivity that results in the appearance or disappearance of immunoreactive bands as noted for anti-F-LDN-F and LDN-F.

At present the mechanism(s) by which LTPs alter plasma protein glycotope reactivity is not precisely known. However, two of the most likely possibilities include (1) the binding of LTP directly to subsets of blotted plasma proteins that results in either enhanced glycotope display if the bound LTP are glycoproteins, or decreased glycotope reactivity due to LTP binding and blocking mAB-reactive plasma glycans, or (2) the presence of specific glycosidases in LTP preparations that are capable of enzymatically altering glycan structures, thereby potentially changing the pattern of specific glycotope reactivity displayed by plasma proteins. A proteomic analysis of *S. mansoni* LTP recently identified an α-N-acetylgalactosaminidase and an endo-α-mannosidase [@pntd.0001569-Wu1], both of which may have potential for altering or removing non-substituted GalNAc (as part of LDN, LDN-F and possibly LDN-DF) and TriMan structures, destroying their mAB-reactivities in the process. However, although specific glycosidase activities may account for LTP-associated decreases in plasma glycotope reactivity, their presence/activities cannot explain why exposure to LTP often results in enhanced mAB reactivity for selected plasma proteins (e.g., LDN, LDN-F) or variable reactivity for a specific mAB among different proteins in the same plasma sample (e.g., F-LDN-F). Moreover, because the endo-α-mannosidase (pH optimum, 7.0) only cleaves α1--2 linkages of extended oligomannose structures [@pntd.0001569-Lubas1], it is highly unlikely to be active against the α1--3 and α1--6 linkages comprising the TriMan N-glycan core. The *S. mansoni* α-N-acetylgalactosaminidase also is not predicted to cleave the β1--4 linkage of LDN, thereby rendering it incapable of altering LDN-associated glycotopes. Finally the pH optimum for this enzyme is between 4.3--4.8 [@pntd.0001569-Dean1] and likely would not be active at the pH of the LTP incubation medium (pH 7.4). Therefore, based on the current data, we hypothesize that the direct binding of specific LTPs to selected plasma proteins is responsible for the observed changes in plasma glycotope staining patterns. At this point, however, we do not know to what extent this LTP-plasma binding is due to protein-CHO or protein-protein interactions. Because LTPs appear to bind selectively to subsets of plasma proteins, and in many cases resulting in significant increases or decreases in shared glycotopes associated with a LTP-protein complex, one can imagine a scenario in which miracidia undergoing transformation within the snail host release LTPs that selectively bind to plasma proteins that may be serving as PRRs. Indeed, many of the changes in plasma immunoreactivity seen in LTP-exposed blots occur in proteins between 50--100 kDa (e.g., LDN, LDN-F, F-LDN-F, LDN-DF), which corresponds generally to the molecular size range of the major *B. glabrata* Freps [@pntd.0001569-Adema2]. Current efforts are being focused on identification of the host hemolymph proteins involved in LTP binding interactions.

The findings reported here, and in other recent investigations (reviewed in [@pntd.0001569-Bayne1]--[@pntd.0001569-Mitta1]) present a number of unanswered questions regarding the functional significance of glycans shared between and/or manipulated within schistosome-snail systems. According to the compatibility polymorphism hypothesis detailed by Mitta et al. [@pntd.0001569-Mitta1], the maintenance of high molecular polymorphism (genetic diversity) in both host PRRs (e.g., lectin-like proteins) and their corresponding parasite PAMPs (e.g., glycan-bearing polymorphic mucins) may serve as a possible co-evolutionary mechanism driving the compatibility/incompatibility phenotypes observed in this, and other snail-trematode systems. Although the mechanisms by which snail hemocytes first recognize invading schistosome larvae (including both cellular attraction and adhesion/encapsulation) are still unknown, members of a highly diversified family of fibrinogen-related proteins (Freps) have been implicated as anti-trematode PRRs [@pntd.0001569-Hanington1], [@pntd.0001569-Hanington2], [@pntd.0001569-Zhang1]. Since it has been shown that lectin-like Freps utilize sugars as recognition ligands [@pntd.0001569-Hanington1], glycan structures naturally shared between larval *S. mansoni* and host hemolymph or parasite molecules that alter the composition of native glycans displayed by plasma glycoproteins may serve an important role in determining the reactivity of hemocytes towards an individual parasite developing within a given host [@pntd.0001569-Bayne1], [@pntd.0001569-Yoshino2], [@pntd.0001569-Yoshino3].

With reference to the parasite, genes encoding the polymorphic mucin family (*Sm*PoMuc; [@pntd.0001569-Roger1], [@pntd.0001569-Roger2]) are proposed to provide sufficient genetic diversity to maintain an adequate counterbalance to the highly diversified snail Freps [@pntd.0001569-Mitta1]. This is a reasonable notion, especially when one takes into account the added ligand diversity provided by the O- and N-linked glycan structures that can vary greatly, both qualitatively and quantitatively [@pntd.0001569-Peterson1], [@pntd.0001569-Lehr3]. This glycan diversity was well illustrated in our study showing that, not only are parasite glycans (glycotopes) naturally found on numerous plasma proteins, but snails differing in their susceptibility to the *S. mansoni* strain used in this study, also differed in their repertoires of plasma-associated glycans (e.g., LDN and F-LDN). Moreover, snail-strain differences in the distribution of shared glycans also were noted following LTP treatment of plasma blots (e.g., LeX, F-LDN, LDN-F, LDN-DF).

What is emerging from the present, and other related, investigations is a complex picture of host-parasite molecular interactions that, in accordance with the compatibility polymorphism hypothesis, should provide important clues as to which combinations of host immune factors and corresponding larval factors determine infection success or failure. In support of the above hypothesis, Mone et al. [@pntd.0001569-Mone1] found that fibrinogen-related protein Frep2 bound *Sm*PoMuc in a coimmunoprecipitated complex, which also included, among other adhesion molecules, a Gal-binding lectin. Identifying the repertoire of host immune PRRs (specifically in cell-free plasma and circulating hemocytes) and their specific target ligands (tegumental surface- and LTP-associated glycans) represents the next critical step towards gaining a more comprehensive understanding of the molecular basis for compatibility and incompatibility in this laboratory model of molluscan schistosomiasis. As in mammalian host immune responses to schistosomes [@pntd.0001569-Hokke2], [@pntd.0001569-vanDie1], it is predicted that glycans, especially those shared with the host, will be shown to play prominent roles as modulators and/or mediators of larval-snail immune interactions.

We wish to thank Mike Lindeke and Mike Gehring for maintaining snail colonies. Schistosome-infected mice and uninfected *B. glabrata* were supplied by Dr. Fred Lewis (Biomedical Research Institute, Rockville, MD) under NIH-NIAID Contract No. HHSN272201000005I.

The authors have declared that no competing interests exist.

This work was supported by National Institutes of Health grant no. 2RO1AI015503 to TPY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[^1]: Conceived and designed the experiments: TPY X-JW CHH. Performed the experiments: X-JW HL LAG. Analyzed the data: TPY X-JW AMD CHH. Contributed reagents/materials/analysis tools: AMD CHH. Wrote the paper: TPY. Reviewed/revised manuscript: LAG X-JW CHH AMD. Prepared figures: X-JW LAG. Financial support: TPY.