Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Jan 1.
Published in final edited form as: Fish Shellfish Immunol. 2017 Oct 28;72:111–116. doi: 10.1016/j.fsi.2017.10.037

Allelic variation partially regulates galactose-dependent hydrogen peroxide release from circulating hemocytes of the snail Biomphalaria glabrata

Euan R O Allan 1,*, Michael S Blouin 1
PMCID: PMC5732054  NIHMSID: NIHMS917827  PMID: 29107743

Abstract

Freshwater snails are the intermediate hosts for numerous parasitic worms that are detrimental to human and agricultural health. Understanding the immune responses of these snails could be vital for finding ways to block transmission of those parasites. Allelic variation in a recently discovered genomic region in the snail, Biomphalaria glabrata, influences their susceptibility to schistosomes. Here we tested whether genes in that region, termed the Guadeloupe Resistance Complex (GRC), are involved in recognition of common pathogen-associated molecules that have been shown to be stimulants of the hydrogen peroxide defense pathway. We show that hemocytes extracted from individuals with one of the three GRC genotypes released less hydrogen peroxide than the other two genotypes, after stimulation with galactose. This difference was not observed after stimulation with several other microbial-associated carbohydrates, despite those ligands sharing the same putative pathway for hydrogen peroxide release. Therefore, we conclude that allelic variation in the GRC region may influence the recognition of galactose, rather than the conserved downstream steps in the hydrogen peroxide pathway. These results thus are consistent with the hypothesis that proteins produced by this region are involved in pathogen recognition.

Keywords: Biomphalaria glabrata, innate immunity, carbohydrate, hydrogen peroxide, hemocyte

1. Introduction

Gastropod mollusks are essential for the transmission of many mammalian parasites which are detrimental to human health and agricultural productivity (e.g. blood flukes (Schistosoma), liver flukes (such as Fasciola and Chlonorchis), intestinal flukes (Echinostoma), lungworms (Angiostrongylus) etc.) [1, 2]. Trematodes are the most important class of parasites transmitted by mollusks, and are spread by snails from the families Pomatiopsidae and Planorbidae [2–4]. Understanding the immunological responses of these molluscan hosts may inform on the pathways or mechanisms by which snail species stave off infections, and thus potentially lead to new ways to block transmission of these diseases.

Biomphalaria glabrata is a prominent intermediate host for the human pathogen Schistosoma mansoni in the Americas, making it an invertebrate of intense human interest [4, 5]. Allelic variation at a recently discovered genomic region in Guadeloupean B. glabrata (BgGUA), termed the Guadeloupe Resistance Complex (GRC), has been shown to have a very strong effect on the resistance of BgGUA to Guadeloupean S. mansoni (SmGUA) [6]. Also, novel transmembrane proteins in this region have been shown to influence schistosome infectivity for snails [7]. Although the putative structure of these proteins strongly suggest they are involved in pathogen recognition [6, 7], to date we have no experimental evidence of that hypothesis.

There are three highly divergent haplotypes of the GRC region that show the statistical signature of being under long-term balancing selection [6]. Schistosomes were introduced to the Americas relatively recently, so immune pathways that are associated with resistance almost certainly evolved to combat other pathogens long before schistosomes were introduced. Additionally, there are 7 proteins in the GRC which appear to be structurally similar to receptors because they are predicted to be membrane spanning with hypervariable extracellular domains [6, 7]. Therefore, we hypothesize that the three alleles in the GRC region have been maintained by balancing selection via their differential abilities to recognize various pathogens, and that one of the three alleles just happens to recognize schistosomes more strongly than the other two. Under that scenario, the proteins coded for in this region could function as pattern recognition receptors which bind to conserved pathogen associated molecular patterns (PAMPs) exposed on the surface of invading microbes. Therefore, we tested whether different genotypes of the GRC region differentially recognize a variety of common PAMPs, as assayed by the release of hydrogen peroxide by hemocytes of each genotype after exposure to these PAMPs.

Receptor mediated phagocytosis, and the reactive oxygen species (ROS) pathway terminating with hydrogen peroxide production, are a vital process in innate immune defense for most organisms [8, 9]. Intraphagosmal ROS, and the ensuing extracellular hydrogen peroxide that is released following exposure to PAMPs, are a key component of the defensive pathway in B. glabrata [10–15]. The cascade of events preceding canonical hydrogen peroxide production in hemocytes begins with pattern receptors binding a PAMP, and the subsequent phagocytosis of the bound microbial product (Figure 1). This is immediately followed by NADPH oxidase 2 (NOX2) recruitment to the phagosomal membrane and intraphagosmal superoxide production [8, 16, 17]. This reactive mediator is used as a substrate for cytosolic superoxide dismutases (SODs) to produce extracellular hydrogen peroxide, which is one of the primary mediators of oxidative damage to extracellular pathogens [9]. Many microbes exhibit carbohydrates on their surface which are recognized by hemocytes as a PAMP and can induce the microbicidal receptor mediated canonical hydrogen peroxide production pathway (Figure 1) [12, 15, 18, 19]. Despite the importance of these PAMPs, there is little known about molluscan-specific carbohydrate-binding pattern recognition pathways. Characterizing the genes that influence these responses is essential for fully understanding molluscan immunity.

Fig 1. A simplified diagram outlining the known canonical hydrogen peroxide pathway in B. glabrata.

Fig 1

Open in a new tab

Both PMA, and ligands that require a receptor on the cell surface, activate intracellular kinases before NOX2 is recruited and activated. NOX2 produces superoxide (O2−), the substrate for SOD, which is then converted to hydrogen peroxide. Hydrogen peroxide acts as the final anti-microbial product in this pathway in hemocytes. The kinases and receptors which lead to the activation of NOX2 are not conclusively characterized in B. glabrata, though the downstream NOX2-SOD pathway is believed to be conserved with that of mammals. The receptor initiating the hemocyte response is likely to be the primary difference between stimulation with the ligands used in this study (other than PMA).

Here we show that allelic variation in the GRC has an effect on the release of hydrogen peroxide by hemocytes after stimulation with galactose, but not after stimulation with other PAMPs that should induce the same conserved ROS pathway. This result suggests that the allelic variation in the GRC acts very early in the ROS pathway, potentially at the recognition stage. These findings further implicate this genomic region in molluscan immune defense.

2. Materials and Methods

2.1 Animals and ethics

BgGUA and SmGUA were collected in 2005 from the island of Guadeloupe, and maintained/cycled under standard conditions as previously described [6, 20, 21]. BgGUA snails were genotyped at the GRC locus, and segregated into 9 independent lines as previously described: 3 RR (resistant lines R1, R2, R3), 3 S1S1 (susceptible lines S11, S12, S13), and 3 S2S2 (susceptible lines S21, S22, S23) [6]. For nearly all experiments equal numbers of snails were pooled from each of the three lines within a GRC genotype (RR, S1S1, S2S2), and in one experiment all the individual lines were tested. All animals were housed identically and age matched (9–15 mm). All experiments adhere to the Public Health Service Domestic Assurance for humane care and use of laboratory animals (PHS Animal Welfare Assurance Number A3229-01), as Animal Care and Use Proposal 4360. This protocol was approved by the Oregon State University Institutional Animal Care and Use Committee.

2.2 BgGUA hemolymph collection, hemocyte isolation and stimulation, and hydrogen peroxide production

We examined hydrogen peroxide release from stimulated hemocytes as an output measure of PAMP stimulation/recognition. To do this, hemolymph was collected from age matched BgGUA by a combination of the headfoot retraction technique and cardiac puncture [10, 12, 15, 22]. In brief, snails were cleaned with 70% ethanol and bled using headfoot retraction followed by a cardiac puncture. Unless otherwise specified, hemolymph from 3 lines of each RR, S1S1, S2S2 was pooled by genotype on parafilm. Shell debris was allowed to settle, cells for each pooled group were counted (enumerated using a hemocytometer) and diluted with CBSS to an equal cell concentration, and 100 μl aliquots of hemolymph were allowed to settle in sterile 96-well black-walled/clear-bottom culture plates (Corning) pre-incubated with 100 μl of 26 °C CBSS/well. After 100 minutes, and 3 successive 15 minute washes with 200 μl of CBSS, the CBSS was removed and replaced with 100 μl of CBSS or CBSS containing pre-established concentrations of one of the following: phorbol 12-myristate 13-acetate (PMA: 500 nM [10, 15]), Diphenyleneiodonium (DPI: 0.5 μM [16]), Lipopolysaccharide from E. coli (LPS: 100 μg/ml [23]), bovine serum albumin (BSA)-conjugated sugars: BSA-mannose (200 nM [12]), BSA-fructose (200 nM [12]), BSA-arabanose (200 nM [24]), BSA-sialic acid (300 nM [25]), and BSA-galactose (300 nM [12, 15]). All compounds were purchased from Sigma-Aldrich (St. Louis, MO), and all BSA conjugated sugars were purchased from Vector Labs (Burlingame, CA). An initial dose response was also conducted using increasing concentrations of each compound using RR hemolymph. After 2 h the hydrogen peroxide (a proxy measure of the respiratory burst) was assessed by the Amplex Red Hydrogen Peroxide Assay Kit (Molecular Probes) according to the manufacturer’s instructions (30 min post exposure to Amplex Red reagent) [10, 15, 26–28]. PMA (+ control, causes endogenous superoxide production) and DPI (−control, direct inhibitor of NOX2) were used as positive and negative controls for bursting cells. All experiments are shown as relative fluorescent units (RFU)/5000 cells, and made relative to the S1S1, or normalized to the PMA control where indicated. Normalizing to the PMA control, within each genotype, was done to control for the number of cells experiencing a respiratory burst. All experiments were conducted at 26 °C using a Molecular Devices Gemini XS Fluorescent Microplate Reader.

2.3 Statistical analyses

Statistical analyses were completed by one-way ANOVA with a Tukey post-test unless otherwise specified (p<0.05). If a Barlett’s test for equal variance failed, then data underwent a natural log transformation (ln). All analyses were completed using GraphPad Prism software (La Jolla, CA, USA).

3. Results

3.1 BgGUA hemocytes produce hydrogen peroxide in response to PMA and microbial products

As has been shown by numerous groups in multiple organismal phyla, PMA and DPI respectively induce, and inhibit, the production of hydrogen peroxide by phagocytic immune cells (Figure 1, Figure 2a) [10, 15, 16, 29, 30]. Using PMA as a positive control for the number of hydrogen peroxide producing cells, we examined up to 4 doses of other stimulants of hydrogen peroxide (Figure 2b). As expected, different microbial products and BSA-conjugated carbohydrates induced the production of varying amounts of hydrogen peroxide by hemocytes and LPS exhibits low levels of stimulation. We verify that mannose and galactose exposure result in large increases in hydrogen peroxide production by hemocytes (Figure 2b). These are some of the most common carbohydrates on the surface of microbes, and have been shown to elicit strong responses by B. glabrata hemocytes [12, 15]. The concentrations of microbial product or BSA-conjugated carbohydrates that elicited the greatest amount of non-fatal hydrogen (no change in live cell count) peroxide production were used in subsequent experiments (Figure 2b) [10, 12, 15, 16, 23–25, 31].

Fig 2. BgGUA hemocytes produce hydrogen peroxide in response to PMA, LPS, and microbial-associated BSA-conjugated sugars.

Fig 2

Open in a new tab

(A) Stimulation of BgGUA hemocytes with PMA (500 nM), PMA (500 nM) + DPI (0.5 μM), or CBSS carrier alone (n=4). (B) A dose response of BgGUA hemocytes to sugars commonly found on microbes, and microbial products isolated directly from microbes. Doses 0–4 correspond to: LPS 0, 10, 50, 100, 150 μg/ml; BSA-sugars 0, 50, 100, 200, 300 nM (n=1 per treatment). All data are shown as RFU/5000 cells relative to the PMA + control. Data in (A) are presented as mean +/− SD relative to PMA stimulation. Significant differences (ANOVA p<0.05) in (A) are denoted by an asterisks (*).

3.2 Allelic variation in the GRC region does not affect hydrogen peroxide production by hemocytes after stimulation with PMA or bacterial LPS

PMA has been shown to induce canonical hydrogen peroxide production by innate immune cells by initiating the recruitment of NOX2 though protein kinase C and mitogen-activated protein kinases (Figure 1) [15, 30]. There appears to be no general dysfunction, as a result of allelic differences in the GRC region, in the hydrogen peroxide production pathway of BgGUA hemocytes. Additionally, receptor mediated hydrogen peroxide production by bacterial LPS was also unaffected by hemocyte genotype (Figure 3). Consequently, the canonical pathway induced by PMA, or via a classical PAMP, was unaffected by allelic variation in the GRC region.

Fig 3. Allelic variation at the GRC locus does not modify hydrogen peroxide stimulation induced by PMA or LPS.

Fig 3

Open in a new tab

Stimulation of RR, S1S1, or S2S2 BgGUA hemocytes with (A) PMA (500 nM), or (B) LPS (100 μg/ml). All data are shown as mean +/− SD of RFU/5000 cells. PMA data (A) are presented relative to the S1S1 genotype (n=4). Data in (B) are normalized to independent PMA + controls (500 nM) within an individual genotype and presented relative to PMA (n=4). Significant differences between genotypes (ANOVA p<0.05) are denoted by an asterisks (*).

3.3 Allelic variation in the GRC region influences galactose-dependent hydrogen peroxide production

Hemocytes of the three genotypes exhibit similar responses to 6 different ligands (Figure 4a–d). However, S2S2 hemocytes produce significantly less hydrogen peroxide (~40–45 % less), than S1S1 or RR hemocytes when exposed to BSA-conjugated galactose (Figure 4e). This phenotype was also observed in multiple, independent, inbred snail lines that are homozygous for each genotype (Figure 4f). All responses are presented relative to the PMA positive control (Figure 4a,e–f), and all ligands caused the release of hydrogen peroxide from hemocytes.

Fig 4. S2S2 hemocytes release less hydrogen peroxide in response to BSA-galactose, but not in response to other common microbial carbohydrates.

Fig 4

Open in a new tab

Stimulation of RR, S1S1, or S2S2 BgGUA hemocytes with (A) BSA-mannose (200 nM), (B) BSA-fructose (200 nM), (C) BSA-arabanose (200 nM), (D) BSA-sialic acid (300 nM), (E) BSA-galactose (300 nM) (n=4). (F) Additionally, three individual families each of RR, S1S1, or S2S2 were independently tested with BSA-galactose (300 nM) (n=4–5). All experiments contained internal PMA + controls (500 nM) within an individual genotype or line. All data are shown as mean +/− SD of RFU/5000 cells, normalized to PMA + controls within an individual genotype, and presented relative to PMA stimulation. Significant differences (ANOVA p<0.05) are denoted by an asterisks (*).

4. Discussion

Galactose is a monosaccharide associated with the cell surface of parasites, yeasts and bacterial pathogens [12, 15, 32, 33]. Additionally it has been shown to elicit powerful oxidative bursts by hemocytes, presumably initiated by lectin-type receptors [12, 15]. Our findings show that hydrogen peroxide production by hemocytes, in response to BSA-galactose, is partially dependent on allelic variation in the GRC because hemocytes from S2S2 individuals produced less than 60% of the hydrogen peroxide of hemocytes of the other two genotypes.

Microbial glycans containing galactose, mannose, and fructose have been shown to induce hemocyte-mediated hydrogen peroxide production as a potential defensive response in B. glabrata [12, 15]. These responses are believed to be the result of lectins, specific carbohydrate-binding proteins. Lectins can recognise microbial derived carbohydrates, bind to lectin specific receptors on the surface of innate immune cells, and potentiate immune responses against those microbes including the production of hydrogen peroxide [2, 4, 18, 19, 34, 35]. The canonical hydrogen peroxide production induced by PMA, bacterial LPS, and 4 pathogen-associated carbohydrates (including mannose and fructose) was unaffected by allelic variation in the GRC region. Although the pathway leading to the activation of SOD enzymes can potentially differ between cell mitogens (PMA) and receptor induced stimulation, the receptor-induced SOD pathway is believed to be relatively conserved in most species and requires the production of oxidative radicals by NOX2 activation to precede SOD activity (Figure 1) [8, 15, 36, 37]. Our findings indicate that it is extremely unlikely that allelic variation in the GRC perturbs any part of hemocytes’ intracellular hydrogen peroxide pathway, because most known SOD inducing stimuli potentiate equivalent hemocyte responses across the GRC genotypes. Therefore, we theorize that one or more proteins in the GRC act at the receptor level to regulate carbohydrate/lectin dependent immune response in hemocytes. Given that S1S1 and S2S2 individuals have equivalent rates of infection [6, 7] but differential ROS production, and that ROS are integral to schistosome clearance, it is unlikely resistant BgGUA are recognising SmGUA via galactose-containing glycoproteins. Future work could use this redox-sensitive assay to examine additional schistosome products, with the goal of determining if any produce more ROS in RR BgGUA when compared to SS BgGUA.

In summary, the GRC region can modify the release of hydrogen peroxide from hemocytes after they are stimulated with BSA-galactose, but this same allelic variation had no effect on hydrogen peroxide produced after simulation with other BSA-conjugated carbohydrates, LPS, or PMA. Therefore, it is unlikely that proteins encoded in the GRC are involved in critical intracellular steps in the production of hydrogen peroxide, but instead are potentially involved in some part of the recognition of glycoproteins containing galactose. This speculation regarding PAMP recognition is consistent with the putative structure of many proteins in the GRC region. Seven of the GRC proteins are predicted to have conserved intracellular and transmembrane domains, while also expressing hypervariable extracellular domains which are reminiscent of transmembrane receptors [6, 7]. Thus, our results are consistent with the hypothesis that some protein/s in the GRC region may be involved in pathogen recognition in snails.

The present study expands the basic knowledge regarding the immune responses of snails to microbial stimuli. We have shown that the GRC region is involved in galactose-dependent hydrogen peroxide production, potentially via lectin-type PAMP recognition/responses. Given that the GRC region is likely to have an important role in molluscan immunity to numerous microbes, continued investigation into the immunological roles of proteins in this region is warranted.

Highlights.

  • *

    The Guadeloupe Resistance complex is potentially involved in galactose recognition

  • *

    The Guadeloupe Resistance complex is involved in the production of hydrogen peroxide

  • *

    The Guadeloupe Resistance complex may have multiple immune roles

Acknowledgments

We would like to thank Christopher Bayne, Jacob Tennessen, Clint Sergi, Stephanie Bollmann, Leeah Whittier, Ryan Wilson, and Ekaterina Peremyslova for their technical support and advice. Funding: This work was supported by the National Institutes of Health [AI109134 and AI111201].

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Giannelli A, Cantacessi C, Colella V, Dantas-Torres F, Otranto D. Gastropod-Borne Helminths: A Look at the Snail-Parasite Interplay. Trends Parasitol. 2016;32(3):255–64. doi: 10.1016/j.pt.2015.12.002. [DOI] [PubMed] [Google Scholar]
  • 2.Loker ES. Gastropod immunobiology. Adv Exp Med Biol. 2010;708:17–43. doi: 10.1007/978-1-4419-8059-5_2. [DOI] [PubMed] [Google Scholar]
  • 3.Coustau C, Gourbal B, Duval D, Yoshino TP, Adema CM, Mitta G. Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade. Fish Shellfish Immunol. 2015;46(1):5–16. doi: 10.1016/j.fsi.2015.01.036. [DOI] [PubMed] [Google Scholar]
  • 4.Pearce EJ, MacDonald AS. The immunobiology of schistosomiasis. Nat Rev Immunol. 2002;2(7):499–511. doi: 10.1038/nri843. [DOI] [PubMed] [Google Scholar]
  • 5.Reardon S. Welcome to the CRISPR zoo. Nature. 2016;531(7593):160–3. doi: 10.1038/531160a. [DOI] [PubMed] [Google Scholar]
  • 6.Tennessen JA, Theron A, Marine M, Yeh JY, Rognon A, Blouin MS. Hyperdiverse gene cluster in snail host conveys resistance to human schistosome parasites. PLoS Genet. 2015;11(3):e1005067. doi: 10.1371/journal.pgen.1005067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Allan ER, Tennessen JA, Bollmann SR, Hanington PC, Bayne CJ, Blouin MS. Schistosome infectivity in the snail, Biomphalaria glabrata, is partially dependent on the expression of Grctm6, a Guadeloupe Resistance Complex protein. PLoS Negl Trop Dis. 2017;11(2):e0005362. doi: 10.1371/journal.pntd.0005362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87(1):245–313. doi: 10.1152/physrev.00044.2005. [DOI] [PubMed] [Google Scholar]
  • 9.Fukai T, Ushio-Fukai M. Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal. 2011;15(6):1583–606. doi: 10.1089/ars.2011.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bender RC, Broderick EJ, Goodall CP, Bayne CJ. Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails. J Parasitol. 2005;91(2):275–9. doi: 10.1645/GE-415R. [DOI] [PubMed] [Google Scholar]
  • 11.Goodall CP, Bender RC, Brooks JK, Bayne CJ. Biomphalaria glabrata cytosolic copper/zinc superoxide dismutase (SOD1) gene: association of SOD1 alleles with resistance/susceptibility to Schistosoma mansoni. Mol Biochem Parasitol. 2006;147(2):207–10. doi: 10.1016/j.molbiopara.2006.02.009. [DOI] [PubMed] [Google Scholar]
  • 12.Hahn UK, Bender RC, Bayne CJ. Production of reactive oxygen species by hemocytes of Biomphalaria glabrata: carbohydrate-specific stimulation. Dev Comp Immunol. 2000;24(6–7):531–41. doi: 10.1016/s0145-305x(00)00017-3. [DOI] [PubMed] [Google Scholar]
  • 13.Hahn UK, Bender RC, Bayne CJ. Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata. J Parasitol. 2001;87(4):778–85. doi: 10.1645/0022-3395(2001)087[0778:IONOIK]2.0.CO;2. [DOI] [PubMed] [Google Scholar]
  • 14.Hahn UK, Bender RC, Bayne CJ. Killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata: role of reactive oxygen species. J Parasitol. 2001;87(2):292–9. doi: 10.1645/0022-3395(2001)087[0292:KOSMSB]2.0.CO;2. [DOI] [PubMed] [Google Scholar]
  • 15.Humphries JE, Yoshino TP. Regulation of hydrogen peroxide release in circulating hemocytes of the planorbid snail Biomphalaria glabrata. Dev Comp Immunol. 2008;32(5):554–62. doi: 10.1016/j.dci.2007.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Balce DR, Li B, Allan ER, Rybicka JM, Krohn RM, Yates RM. Alternative activation of macrophages by IL-4 enhances the proteolytic capacity of their phagosomes through synergistic mechanisms. Blood. 2011;118(15):4199–208. doi: 10.1182/blood-2011-01-328906. [DOI] [PubMed] [Google Scholar]
  • 17.Rybicka JM, Balce DR, Chaudhuri S, Allan ER, Yates RM. Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner. EMBO J. 2012;31(4):932–44. doi: 10.1038/emboj.2011.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Esko JD, Sharon N. Microbial Lectins: Hemagglutinins, Adhesins, and Toxins. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. Cold Spring Harbor; NY: 2009. [PubMed] [Google Scholar]
  • 19.Lejoly-Boisseau H, Appriou M, Seigneur M, Pruvost A, Tribouley-Duret J, Tribouley J. Schistosoma mansoni: in vitro adhesion of parasite eggs to the vascular endothelium. Subsequent inhibition by a monoclonal antibody directed to a carbohydrate epitope. Exp Parasitol. 1999;91(1):20–9. doi: 10.1006/expr.1999.4348. [DOI] [PubMed] [Google Scholar]
  • 20.Theron A, Rognon A, Gourbal B, Mitta G. Multi-parasite host susceptibility and multi-host parasite infectivity: a new approach of the Biomphalaria glabrata/Schistosoma mansoni compatibility polymorphism. Infect Genet Evol. 2014;26:80–8. doi: 10.1016/j.meegid.2014.04.025. [DOI] [PubMed] [Google Scholar]
  • 21.Theron A, Coustau C, Rognon A, Gourbiere S, Blouin MS. Effects of laboratory culture on compatibility between snails and schistosomes. Parasitology. 2008;135(10):1179–88. doi: 10.1017/S0031182008004745. [DOI] [PubMed] [Google Scholar]
  • 22.Sminia T, Barendsen L. A Comparative morphological and enzyme histochemical study on blood cells of the fresh-water snails Lymnaea stagnalis, Biomphalaria glabrata, and Bulinus truncatus. J Morphol. 1980;165(1):31–39. doi: 10.1002/jmor.1051650104. [DOI] [PubMed] [Google Scholar]
  • 23.Soldatos AN, Metheniti A, Mamali I, Lambropoulou M, Marmaras VJ. Distinct LPS-induced signals regulate LPS uptake and morphological changes in medfly hemocytes. Insect Biochem Mol Biol. 2003;33(11):1075–84. doi: 10.1016/s0965-1748(03)00119-x. [DOI] [PubMed] [Google Scholar]
  • 24.Bahat-Samet E, Castro-Sowinski S, Okon Y. Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense. FEMS Microbiol Lett. 2004;237(2):195–203. doi: 10.1016/j.femsle.2004.06.036. [DOI] [PubMed] [Google Scholar]
  • 25.Varki A, Gagneux P. Multifarious roles of sialic acids in immunity. Ann N Y Acad Sci. 2012;1253:16–36. doi: 10.1111/j.1749-6632.2012.06517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Allan ER, Tailor P, Balce DR, Pirzadeh P, McKenna NT, Renaux B, Warren AL, Jirik FR, Yates RM. NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing. J Immunol. 2014;192(11):4989–5001. doi: 10.4049/jimmunol.1302896. [DOI] [PubMed] [Google Scholar]
  • 27.Allan ER, Yates RM. Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis. PLoS One. 2015;10(6):e0128945. doi: 10.1371/journal.pone.0128945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Allan ERO, Campden RI, Ewanchuk BW, Tailor P, Balce DR, McKenna NT, Greene CJ, Warren AL, Reinheckel T, Yates RM. A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis. J Neuroinflammation. 2017;14(1):103. doi: 10.1186/s12974-017-0874-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Donaghy L, Kraffe E, Le Goic N, Lambert C, Volety AK, Soudant P. Reactive oxygen species in unstimulated hemocytes of the pacific oyster Crassostrea gigas: a mitochondrial involvement. PLoS One. 2012;7(10):e46594. doi: 10.1371/journal.pone.0046594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Liles WC, Ledbetter JA, Waltersdorph AW, Klebanoff SJ. Cross-linking of CD45 enhances activation of the respiratory burst in response to specific stimuli in human phagocytes. J Immunol. 1995;155(4):2175–84. [PubMed] [Google Scholar]
  • 31.Humphries JE, Yoshino TP. Schistosoma mansoni excretory-secretory products stimulate a p38 signalling pathway in Biomphalaria glabrata embryonic cells. Int J Parasitol. 2006;36(1):37–46. doi: 10.1016/j.ijpara.2005.08.009. [DOI] [PubMed] [Google Scholar]
  • 32.Vazquez A, Ruiz-Rosado JD, Terrazas LI, Juarez I, Gomez-Garcia L, Calleja E, Camacho G, Chavez A, Romero M, Rodriguez T, Espinoza B, Rodriguez-Sosa M. Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection. Int J Biol Sci. 2014;10(8):909–920. doi: 10.7150/ijbs.9214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Peng XA, Sun J, Michiels C, Iserentant D, Verachtert H. Decrease in cell surface galactose residues of Schizosaccharomyces pombe enhances its coflocculation with Pediococcus damnosus. Appl Environ Microb. 2001;67(8):3413–3417. doi: 10.1128/AEM.67.8.3413-3417.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Hanington PC, Forys MA, Dragoo JW, Zhang SM, Adema CM, Loker ES. Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection. Proc Natl Acad Sci U S A. 2010;107(49):21087–92. doi: 10.1073/pnas.1011242107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Jiang Y, Loker ES, Zhang SM. In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference. Dev Comp Immunol. 2006;30(10):855–66. doi: 10.1016/j.dci.2005.12.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Abreu IA, Cabelli DE. Superoxide dismutases-a review of the metal-associated mechanistic variations. Bba-Proteins Proteom. 2010;1804(2):263–274. doi: 10.1016/j.bbapap.2009.11.005. [DOI] [PubMed] [Google Scholar]
  • 37.Bonner KM, Bayne CJ, Larson MK, Blouin MS. Effects of Cu/Zn superoxide dismutase (sod1) genotype and genetic background on growth, reproduction and defense in Biomphalaria glabrata. PLoS Negl Trop Dis. 2012;6(6):e1701. doi: 10.1371/journal.pntd.0001701. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES