Schistosomiasis, a disease caused by parasitic trematodes of the genus Schistosoma, is second only to malaria as the most socioeconomically devastating parasitic disease, with an estimated 252 million people infected worldwide in 2015 (Disease et al., 2016; Mortality and GBD 2015 Mortality and Causes of Death Collaborators, 2016). Larval digenean trematodes share a common feature in the use of snails (gastropod mollusks) as intermediate hosts for transmission to a vertebrate host (Esch et al., 2002). The snail Biomphalaria glabrata transmits several species of trematode, including Schistosoma mansoni, the predominant causal species of intestinal schistosomiasis (Pila et al., 2016a). The molecular interactions between B. glabrata and S. mansoni have been studied extensively towards better understanding this essential life cycle stage of an important human parasite (Adema, 2015).

Various genetically determined resistance phenotypes exist with respect to S. mansoni infection in B. glabrata (Richards, 1975a; Richards, 1975b). Some B. glabrata snails (such as BS-90 strain) naturally resist S. mansoni infection. Susceptible B. glabrata (such as M-line strain) can develop transient levels of acquired resistance following previous exposure to incompatible or radiation-attenuated parasites with modest cross protection to related digenean species (Lie and Heyneman, 1975; Lie et al., 1975; Sullivan et al., 1982). Previous research has focused on identifying the mechanisms responsible for determining resistance profiles. Using in vivo and in vitro models of B. glabrata snails, it has been demonstrated that the killing of S. mansoni larvae is associated with a haemocyte-mediated cytotoxic mechanism, and passive transfer of natural resistance to S. mansoni has been successfully accomplished when haemocytes from a susceptible B. glabrata strain are incubated in cell-free haemolymph (plasma) from a resistant strain (Bayne et al., 1980a; Bayne et al., 1980b; Granath and Yoshino, 1984; Loker and Bayne, 1982). Thus, haemocytes from resistant or susceptible B. glabrata strains do not appear to differ a priori in their cytotoxic capabilities, but their response requires activation by some humoral factor(s) (Bayne et al., 1980b; Granath and Yoshino, 1984; Vasquez and Sullivan, 2001), for proper recognition of S. mansoni and enhancing haemocyte cytotoxicity (Hahn et al., 2001).

Researchers have long sought immune determinants present in resistant B. glabrata plasma that specifically activate haemocytes to encapsulate and destroy S. mansoni sporocysts. Identified in B. glabrata, a large family of soluble lectins termed fibrinogen-related proteins (BgFREPs) attracted widespread interest because of their unique structure (Adema et al., 1997; Gordy et al., 2015), capacity for somatic diversification (Zhang et al., 2004), and immune function (Hanington et al., 2010; Hanington and Zhang, 2011). BgFREPs are composed of a C-terminal fibrinogen (FBG) domain and either one or two N-terminal immunoglobulin superfamily (IgSF) domains linked to the FBG domain by an interceding region (ICR) (Léonard et al., 2001; Zhang et al., 2001). Numerous studies have implicated BgFREP3 as playing a central role in B. glabrata resistance to digenetic trematodes (Hanington et al., 2010; Hanington et al., 2012; Hanington and Zhang, 2011; Lockyer et al., 2012; Lockyer et al., 2008; Pila et al., 2017b). BgFREP3 contains two tandem IgSF domains whereas BgFREP2 has one (Léonard et al., 2001). The expression of BgFREP2 in BS-90 snails is dramatically up-regulated (57-fold) after exposure to S. mansoni, whereas M-line snails do not feature such a drastic upregulation, thereby suggesting a role for BgFREP2 in resistance (Hertel et al., 2005). Interestingly, the time course for heightened BgFREP2 expression overlaps the interval within which sporocysts are encapsulated and killed, and only occurs in BS-90 haemocytes (Dinguirard et al., 2018; Hertel et al., 2005). Furthermore it was reported that BgFREP2 is reactive with S. mansoni polymorphic mucins (SmPoMucs) found on the tegumental surface of larval S. mansoni (Moné et al., 2010).

In addition to BgFREPs, other soluble immune effector factors which are involved in both the direct killing of sporocysts and preparation of haemocytes to mount a cell-mediated response have been characterized (Pila et al., 2017a). These factors include B. glabrata thioester-containing proteins (BgTEPs) (Bender et al., 1992; Moné et al., 2010; Portet et al., 2018) and Biomphalysin (Galinier et al., 2013). TEPs are key components of invertebrate and vertebrate immune responses, aiding in melanization, opsonization, and killing of invading pathogens (Baxter et al., 2007; Blandin et al., 2008; Levashina et al., 2001; Pila et al., 2017a; Portet et al., 2018; Povelones et al., 2013; Yassine and Osta, 2010). BgTEP was first characterized as an alpha macroglobulin proteinase inhibitor, and has recently re-emerged as a molecule of interest due to its association with BgFREP2 and SmPoMucs, and the capacity to bind to various invading pathogens (Bender et al., 1992; Moné et al., 2010; Portet et al., 2018). Biomphalysin is a cytolytic protein in B. glabrata belonging to the β pore-forming toxin (β-PFT) superfamily (Galinier et al., 2013). Biomphalysin binds to the surface of S. mansoni sporocysts in the absence of plasma, while its cytolytic activity is drastically increased when plasma is present, suggesting that other factor(s) within the plasma may mediate the conversion of the oligomeric pre-pore to a functional pore (Galinier et al., 2013). Although the functional mechanisms of these factors are not thoroughly understood, studies suggest that these factors function as key determinants in the final outcome of S. mansoni challenge of B. glabrata (Galinier et al., 2013; Moné et al., 2010).

While studies have implicated BgFREP3 in resistance to S. mansoni, the underlying mechanism of BgFREP3 function; how it binds to S. mansoni sporocyst surfaces, and then how recognition is translated into haemocyte engagement, activation, and ultimately parasite encapsulation, is still unknown (Hanington et al., 2010). Here, we report an association between BgFREP3, BgTEP1, Biomphalysin (UniProtKB/TrEMBL: A0A182YTN9 and A0A182YTZ4) and BgFREP2 (UniProtKB/TrEMBL: A0A2C9L9F5). BgFREP3 associates with BgTEP1 and Biomphalysin in both M-line and BS-90 strains, but only in BS-90 strain uniquely interacts with BgFREP2 and other versions of BgFREP3 (a variant of BgFREP3.3), providing us with an important insight into why BS-90 strain is refractory to S. mansoni infection. In this study, we demonstrate that BgFREP3 binds to S. mansoni sporocysts without any other soluble plasma factors, yet binding of BgFREP2 relies on BgTEP1. However, BgFREP3 still interacts with BgTEP1 to form unique immune complexes, which significantly enhance the ability of haemocytes and plasma from S. mansoni-susceptible B. glabrata (M-line) to kill S. mansoni sporocysts. A more striking finding is that the combination of BgFREP3, BgTEP1 and BgFREP2 renders M-line haemocytes capable of killing S. mansoni sporocysts at nearly the same level as S. mansoni-resistant BS-90 haemocytes. Reactive oxygen species (ROS) are shown to play an important role during this haemocyte-mediated killing of S. mansoni sporocysts. These results provide insight into how the numerous previously characterized immune factors known to be important in the anti-S. mansoni immune response to B. glabrata are acting in concert to defend the snail host.

In 1984, Granath and Yoshino successfully achieved passive transfer of resistance to S. mansoni from a refractory B. glabrata strain to susceptible snails via injection of cell-free haemolymph (plasma). Similar passive transfer of resistance had previously been shown using an in vitro model (Bayne et al., 1980a; Bayne et al., 1980b; Loker and Bayne, 1982). Although the oxidation of haemoglobin, which is the main protein component (97% of total protein) in B. glabrata plasma was later found to be toxic to S. mansoni sporocysts in long-term cultures (Bender et al., 2002), and thus likely explained the passive transfer study results, further studies suggest that specific anti-parasitic factors must be present at or above a certain threshold level in S. mansoni-resistant snail plasma to stimulate a haemocyte-mediated destruction of sporocysts within 24–48 hr post-infection (Dinguirard et al., 2018). Although many important immune factors have been identified from B. glabrata plasma/haemocytes, such as BgFREPs (Adema et al., 1997), BgTEP1 (Moné et al., 2010), Biomphalysin (Galinier et al., 2013), Toll-like receptors (BgTLRs) (Pila et al., 2016b), granulin (BgGRN) (Pila et al., 2016a) and macrophage migration inhibitory factor (BgMIF) (Baeza Garcia et al., 2010), it is still not known which factors ultimately underpin B. glabrata immunity to S. mansoni. Here, we provide insight into the broader interactions that take place between B. glabrata immune factors that leads to development of an S. mansoni-resistant phenotype.

Previous studies have shown that BgFREPs are capable of binding to digenean trematode sporocysts and are up-regulated in response to larval infection (Adema et al., 1997; Wu et al., 2017; Zhang et al., 2001). BgFREP3 has been shown to be a central part of the anti-S. mansoni immune response (Hanington et al., 2010). BgFREP3 is up-regulated based on three characteristics (age, strain, and acquired resistance) of resistance of B. glabrata to infection with S. mansoni or Echinostoma paraensei (Hanington et al., 2010). Knock-down of BgFREP3 in resistant snails using siRNA-mediated interference altered the phenotype of resistant B. glabrata by increasing susceptibility to E. paraensei infection (Adema, 2015; Hanington et al., 2010). Nevertheless, we have little knowledge of the underlying mechanism by which BgFREP3 interacts with other snail plasma factors in order to signal and trigger an immune response after recognizing the pathogen. In order to explore this mechanism, we conducted a series of pull-down experiments that allowed us to discover the interaction between BgTEP1, Biomphalysin and BgFREP2 with BgFREP3 (Figure 1).

Proteomic analysis of B. glabrata plasma revealed that BgFREP3, BgTEP1 and BgFREP2 display affinity for S. mansoni sporocyst tegumental membrane proteins (Portet et al., 2018; Wu et al., 2017). BgTEP1 was previously reported to play a role in BgFREP2 recognition of S. mansoni sporocyst SmPoMucs, (Moné et al., 2010; Portet et al., 2018). Our immunofluorescence results illustrate that BgFREP3 independently recognizes S. mansoni sporocysts, whereas BgFREP2 must be involved with the participation of BgTEP1 (Figure 2). We speculate that this may be due to their structural differences: BgFREP3 possesses two IgSF domains, yet BgFREP2 has only one; BgFREP3 exists as a multimer in its natural state (Figure 3), whereas BgFREP2 does not (Zhang et al., 2008). Alternatively, it is possible that the targets they recognize are different, so the immunoaffinity reactions that occur are also fundamentally different. A recent report suggests that BgFREPs are likely to form multimers through the coiled-coil region of the ICR. However, this has not been supported by experimental evidence and does not completely rule out the possibility that BgFREP multimer formation is mediated by the FBG or IgSF domain (Gorbushin, 2019).

BgTEP1 and BgFREP3 form a complex without any S. mansoni molecules present (Figure 3). We do not yet know exactly how BgTEP1 is activated during infection. It is possible that BgFREP3 is stabilizing BgTEP1 like the leucine-rich repeat immune protein (LRIM)/Anopheles Plasmodium-responsive Leucine-rich repeat protein 1 (APL1C) complex that has been found to stabilize TEP in Anopheles gambiae, while also guiding it to the sporocyst’s surface (Fraiture et al., 2009). Additionally, our LC-MS/MS results seem to indicate that BgFREP3 interacted with a full-length form of BgTEP1, while previous work has only found the C-terminal portion of BgTEP1 associating with SmPoMucs (Moné et al., 2010). This may indicate that once brought in association with the parasite, BgTEP1 becomes cleaved/activated. BgFREP3 does not require BgTEP1 to recognize pathogens, this suggests BgTEP1 is more likely to play a role in the downstream immune response initiated by BgFREP3. Following the addition of rBgFREP3 and rBgTEP1, plasma demonstrated an increased capacity to kill sporocysts compared to controls (Figure 4A). Toxicity of haemoglobin oxidation to S. mansoni sporocysts was ruled out as a possible mechanism underpinning sporocyst killing because the B. glabrata plasma used in these tests had haemoglobin removed. These studies also demonstrated that recombinant BgFREP3 and BgTEP1 alone were not able to increase sporocyst killing, but showed that the addition of cell-free plasma with both recombinants was required, thereby implying the necessity of another factor(s). Based on our pull-down experiments, we suggest that this factor could be a Biomphalysin. Biomphalysin possesses many similarities to members of the β-PFT superfamily (Howard and Buckley, 1985; MacKenzie et al., 1999; Wilmsen et al., 1992; Xu et al., 2014), and has been shown to have cytolytic activity that is mediated by a plasma factor(s) that remains to be identified (Galinier et al., 2013). Our results suggest a scenario in which BgFREP3 and/or BgTEP1 could be the factors associating with Biomphalysin to promote plasma-mediated sporocyst killing. BgFREP3 and/or BgTEP1 might activate Biomphalysin in an unknown manner, assist in the oligomerization of Biomphalysin while it forms its heptameric channel, or mediate the conversion of the oligomeric pre-pore to a functional pore. Further investigation is warranted to illustrate the role of BgFREP3 and BgTEP1 in the activation of Biomphalysin.

Within 2–3 hr of S. mansoni entry into B. glabrata, haemocytes surround the developing sporocysts forming multi-layered cellular capsules that kill encapsulated larvae within 24–48 hr post-infection (Dinguirard et al., 2018; Loker and Bayne, 1982). This occurs in both susceptible and resistant snail strains and has been replicated in our in vitro functional studies, where only resistant snails can eventually destroy the sporocysts without external assistance. Dinguirard et al. conducted a comparative proteomic analysis on the responses of haemocytes participating in S. mansoni sporocyst encapsulation reactions from susceptible and resistant B. glabrata strains, showing a striking differences in proteins expressed, with susceptible snail haemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed proteins involved immunity (e.g., BgFREP2) and ROS production compared to resistant snails (Dinguirard et al., 2018). This could explain why BS-90 haemocytes alone can kill S. mansoni sporocysts under normal in vivo conditions. After challenge, the transcript abundance of numerous BgFREPs increases by 3-fold or greater in BS-90 compared to M-line B. glabrata (Adema et al., 1997). Noteworthy in the context of our current study is that following challenge BS-90 snails with S. mansoni, transcript abundance for BgFREP2 increases over 50-fold by 1 day post-exposure (Dinguirard et al., 2018; Hertel et al., 2005), BgFREP3 increases around 4-fold by 1 day post-exposure (Hanington et al., 2010), and BgTEP1 which increases by 2- to 3.5-fold from 6 to 24 hr post-exposure (Portet et al., 2018). These dramatic increases in the abundance of immune-relevant transcripts occurring over the first 12–24 hr post exposure to S. mansoni sporocysts in BS-90 snails aligns closely with our results that demonstrate sporocyst killing by BS-90 haemocytes rapidly increases after 12 hr post-exposure (Figure 4).

Numerous studies have demonstrated the production of ROS by haemocytes in B. glabrata, especially H₂O₂, which plays a vital role in anti-schistosome defense (Galinier et al., 2013; Hahn et al., 2000; Hahn et al., 2001). Haemocytes from S. mansoni-resistant snails produce significantly more ROS than susceptible snails (Bender et al., 2005; Bender et al., 2007; Goodall et al., 2004). In our study, ROS was critical in BS-90 haemocyte-mediated S. mansoni sporocyst killing, as this ability was significantly reduced after pre-incubation with the ROS inhibitors NAC and DPI (Figure 4E). However, according to the phenomenon of passively transferred resistance, the cytotoxic potential of susceptible haemocytes may not be fundamentally different from resistant snails (Granath and Yoshino, 1984). We infer that M-line plasma may lack some factors or may not achieve sufficient thresholds of certain factors to stimulate haemocytes to produce ROS. It is also possible that M-line haemocytes are lacking specific receptors to receive signals that upregulate ROS to effective levels able to kill S. mansoni sporocysts. Previously, our team identified a toll-like receptor (BgTLR) from haemocyte surface of B. glabrata snails, which differed in abundance between M-line and BS-90 strains (Pila et al., 2016b). It has been repeatedly demonstrated in other organisms that activation of Toll-like receptors is associated with increased intracellular ROS (Asehnoune et al., 2004; Tsung et al., 2007; Wong et al., 2009).

Our results indicate that the BgFREP3-BgTEP1 complex plays a role in raising ROS levels in M-line haemocytes (Figure 4F). We found that BS-90 haemolymph contains more BgFREP3 protein than M-line (Figure 1B). Moreover, the BS-90 haemocytes significantly increased the expression level of BgFREP2 during encapsulating S. mansoni sporocysts compared with susceptible snail haemocytes (Dinguirard et al., 2018). These suggest that BgFREP3 and BgFREP2 may play a crucial role in inducing haemocytes which take part in encapsulating sporocysts to generate ROS. We suppose that BgTEP1 also plays an opsonin role in the B. glabrata snail haemolymph, which interacts with molecules on the surface of haemocytes, thereby promoting the phagocytosis of pathogens.

Our data suggests that there are fundamental differences between the BgFREP3-mediated immune response between BS-90 and M-line B. glabrata. Protein Pull-down experiments yielded two obvious and unique protein bands that were present in BS-90 but not M-line pull-downs. We identified the bands to be a variant of BgFREP3.3 and BgFREP2 (Figure 1C), showing different immune factor interactomes between different strains. This suggests that BgFREP3 variants and BgFREP2 in BS-90 plasma are more prone to rBgFREP3 binding, and the combination of different versions of BgFREPs seems to play an important and unknown role in snail resistance. Both BgFREP2 and BgFREP3 are characterized by high diversity, our results imply that the versions of BgFREP2 and BgFREP3 in the two snail strains are different. After exposure to S. mansoni, the expression of BgFREP2 in resistant snails is superior to that of susceptible snails (Dinguirard et al., 2018; Gordy et al., 2015; Hertel et al., 2005). This again suggests that BgFREP2 is important in determining the outcome of the compatibility of B. glabrata against S. mansoni. Our study implies that BgFREP3 seems to play an ‘all or none’ switching effect in determining B.glabrata-S. mansoni compatibility, while BgFREP2 seems to play an additive role.

It has been decades since the discovery and initial characterization of BgFREPs in the B. glabrata immune response to S. mansoni. While numerous studies since have implicated BgFREP3 and BgFREP2 along with a suite of other immune factors, as being important elements of the snail immune response to S. mansoni, how the response is coordinated has remained elusive. Our study presents a model in which many of the known determinants of snail resistance to S. mansoni work in concert to protect the snail host against infection. We map a mechanism for the interaction of BgFREP3, BgTEP1, BgFREP2 and Biomphalysin with S. mansoni sporocysts in B. glabrata snail haemolymph that ultimately leads to sporocyst killing (Figure 5). Central to this response is BgFREP3, which is able to recruit unique representatives of both BgFREP2 and BgFREP3 in S. mansoni-resistant BS-90 B. glabrata. With this new-found understanding of the B. glabrata immune response comes the ability to now undertake comprehensive assessments of how modifications to both the snail host and the parasite influence the underpinning immunological drivers of compatibility in this important model for studying human schistosomes.

Figure 5

Download asset Open asset

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Adema CM
   2. Hertel LA
   3. Miller RD
   4. Loker ES

   (1997) A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection

   PNAS 94:8691–8696.

   https://doi.org/10.1073/pnas.94.16.8691

   • PubMed
   • Google Scholar
2. 1. Adema CM

   (2015) Fibrinogen-Related proteins (FREPs) in mollusks

   Results and Problems in Cell Differentiation 57:111–129.

   https://doi.org/10.1007/978-3-319-20819-0_5

   • PubMed
   • Google Scholar
3. 1. Adema CM
   2. Hillier LW
   3. Jones CS
   4. Loker ES
   5. Knight M
   6. Minx P
   7. Oliveira G
   8. Raghavan N
   9. Shedlock A
   10. do Amaral LR
   11. Arican-Goktas HD
   12. Assis JG
   13. Baba EH
   14. Baron OL
   15. Bayne CJ
   16. Bickham-Wright U
   17. Biggar KK
   18. Blouin M
   19. Bonning BC
   20. Botka C
   21. Bridger JM
   22. Buckley KM
   23. Buddenborg SK
   24. Lima Caldeira R
   25. Carleton J
   26. Carvalho OS
   27. Castillo MG
   28. Chalmers IW
   29. Christensens M
   30. Clifton S
   31. Cosseau C
   32. Coustau C
   33. Cripps RM
   34. Cuesta-Astroz Y
   35. Cummins SF
   36. di Stephano L
   37. Dinguirard N
   38. Duval D
   39. Emrich S
   40. Feschotte C
   41. Feyereisen R
   42. FitzGerald P
   43. Fronick C
   44. Fulton L
   45. Galinier R
   46. Gava SG
   47. Geusz M
   48. Geyer KK
   49. Giraldo-Calderón GI
   50. de Souza Gomes M
   51. Gordy MA
   52. Gourbal B
   53. Grunau C
   54. Hanington PC
   55. Hoffmann KF
   56. Hughes D
   57. Humphries J
   58. Jackson DJ
   59. Jannotti-Passos LK
   60. de Jesus Jeremias W
   61. Jobling S
   62. Kamel B
   63. Kapusta A
   64. Kaur S
   65. Koene JM
   66. Kohn AB
   67. Lawson D
   68. Lawton SP
   69. Liang D
   70. Limpanont Y
   71. Liu S
   72. Lockyer AE
   73. Lovato TL
   74. Ludolf F
   75. Magrini V
   76. McManus DP
   77. Medina M
   78. Misra M
   79. Mitta G
   80. Mkoji GM
   81. Montague MJ
   82. Montelongo C
   83. Moroz LL
   84. Munoz-Torres MC
   85. Niazi U
   86. Noble LR
   87. Oliveira FS
   88. Pais FS
   89. Papenfuss AT
   90. Peace R
   91. Pena JJ
   92. Pila EA
   93. Quelais T
   94. Raney BJ
   95. Rast JP
   96. Rollinson D
   97. Rosse IC
   98. Rotgans B
   99. Routledge EJ
   100. Ryan KM
   101. Scholte LLS
   102. Storey KB
   103. Swain M
   104. Tennessen JA
   105. Tomlinson C
   106. Trujillo DL
   107. Volpi EV
   108. Walker AJ
   109. Wang T
   110. Wannaporn I
   111. Warren WC
   112. Wu XJ
   113. Yoshino TP
   114. Yusuf M
   115. Zhang SM
   116. Zhao M
   117. Wilson RK

   (2017) Whole genome analysis of a schistosomiasis-transmitting freshwater snail

   Nature Communications 8:15451.

   https://doi.org/10.1038/ncomms15451

   • PubMed
   • Google Scholar
4. 1. Asehnoune K
   2. Strassheim D
   3. Mitra S
   4. Kim JY
   5. Abraham E

   (2004) Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B

   The Journal of Immunology 172:2522–2529.

   https://doi.org/10.4049/jimmunol.172.4.2522

   • PubMed
   • Google Scholar
5. 1. Baeza Garcia A
   2. Pierce RJ
   3. Gourbal B
   4. Werkmeister E
   5. Colinet D
   6. Reichhart J-M
   7. Dissous C
   8. Coustau C

   (2010) Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

   PLOS Pathogens 6:e1001115.

   https://doi.org/10.1371/journal.ppat.1001115

   • Google Scholar
6. 1. Baxter RH
   2. Chang CI
   3. Chelliah Y
   4. Blandin S
   5. Levashina EA
   6. Deisenhofer J

   (2007) Structural basis for conserved complement factor-like function in the antimalarial protein TEP1

   PNAS 104:11615–11620.

   https://doi.org/10.1073/pnas.0704967104

   • PubMed
   • Google Scholar
7. 1. Bayne CJ
   2. Buckley PM
   3. DeWan PC

   (1980a) Macrophagelike hemocytes of resistant Biomphalaria glabrata are cytotoxic for sporocysts of Schistosoma mansoni in vitro

   The Journal of Parasitology 66:413–419.

   https://doi.org/10.2307/3280740

   • PubMed
   • Google Scholar
8. 1. Bayne CJ
   2. Buckley PM
   3. DeWan PC

   (1980b) Schistosoma mansoni: cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant Biomphalaria glabrata

   Experimental Parasitology 50:409–416.

   https://doi.org/10.1016/0014-4894(80)90043-0

   • PubMed
   • Google Scholar
9. 1. Bender RC
   2. Fryer SE
   3. Bayne CJ

   (1992) Proteinase inhibitory activity in the plasma of a mollusc: evidence for the presence of α-macroglobulin in Biomphalaria glabrata

   Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 102:821–824.

   https://doi.org/10.1016/0305-0491(92)90086-7

   • Google Scholar
10. 1. Bender RC
    2. Bixler LM
    3. Lerner JP
    4. Bayne CJ

    (2002) Schistosoma mansoni sporocysts in culture: host plasma hemoglobin contributes to in vitro oxidative stress

    Journal of Parasitology 88:14–18.

    https://doi.org/10.1645/0022-3395(2002)088[0014:SMSICH]2.0.CO;2

    • PubMed
    • Google Scholar
11. 1. Bender RC
    2. Broderick EJ
    3. Goodall CP
    4. Bayne CJ

    (2005) Respiratory burst of Biomphalaria glabrata hemocytes: schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails

    The Journal of Parasitology 91:275–279.

    https://doi.org/10.1645/GE-415R

    • PubMed
    • Google Scholar
12. 1. Bender RC
    2. Goodall CP
    3. Blouin MS
    4. Bayne CJ

    (2007) Variation in expression of Biomphalaria glabrata SOD1: a potential controlling factor in susceptibility/resistance to Schistosoma mansoni

    Developmental & Comparative Immunology 31:874–878.

    https://doi.org/10.1016/j.dci.2006.12.005

    • PubMed
    • Google Scholar
13. 1. Blandin SA
    2. Marois E
    3. Levashina EA

    (2008) Antimalarial responses in anopheles gambiae: from a complement-like protein to a complement-like pathway

    Cell Host & Microbe 3:364–374.

    https://doi.org/10.1016/j.chom.2008.05.007

    • PubMed
    • Google Scholar
14. 1. Chernin E

    (1963) Observations on hearts explanted in vitro from the snail australorbis glabratus

    The Journal of Parasitology 49:353–364.

    https://doi.org/10.2307/3275797

    • PubMed
    • Google Scholar
15. 1. Cody JJ
    2. Ittiprasert W
    3. Miller AN
    4. Henein L
    5. Mentink-Kane MM
    6. Hsieh MH

    (2016) The NIH-NIAID schistosomiasis resource center at the biomedical research institute: molecular redux

    PLOS Neglected Tropical Diseases 10:e0005022.

    https://doi.org/10.1371/journal.pntd.0005022

    • PubMed
    • Google Scholar
16. 1. Dinguirard N
    2. Cavalcanti MGS
    3. Wu XJ
    4. Bickham-Wright U
    5. Sabat G
    6. Yoshino TP

    (2018) Proteomic analysis of Biomphalaria glabrata Hemocytes During in vitro Encapsulation of Schistosoma mansoni Sporocysts

    Frontiers in Immunology 9:2773.

    https://doi.org/10.3389/fimmu.2018.02773

    • PubMed
    • Google Scholar
17. 1. Disease GBD
    2. Injury I
    3. Prevalence C
    4. GBD 2015 Disease and Injury Incidence and Prevalence Collaborators

    (2016) Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the global burden of disease study 2015

    Lancet 388:1545–1602.

    https://doi.org/10.1016/S0140-6736(16)31678-6

    • PubMed
    • Google Scholar
18. 1. Esch GW
    2. Barger MA
    3. Fellis KJ

    (2002) The transmission of digenetic Trematodes: style, elegance, complexity

    Integrative and Comparative Biology 42:304–312.

    https://doi.org/10.1093/icb/42.2.304

    • PubMed
    • Google Scholar
19. 1. Fraiture M
    2. Baxter RH
    3. Steinert S
    4. Chelliah Y
    5. Frolet C
    6. Quispe-Tintaya W
    7. Hoffmann JA
    8. Blandin SA
    9. Levashina EA

    (2009) Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of plasmodium

    Cell Host & Microbe 5:273–284.

    https://doi.org/10.1016/j.chom.2009.01.005

    • PubMed
    • Google Scholar
20. 1. Galinier R
    2. Portela J
    3. Moné Y
    4. Allienne JF
    5. Henri H
    6. Delbecq S
    7. Mitta G
    8. Gourbal B
    9. Duval D

    (2013) Biomphalysin, a new β pore-forming toxin involved in Biomphalaria glabrata immune defense against Schistosoma mansoni

    PLOS Pathogens 9:e1003216.

    https://doi.org/10.1371/journal.ppat.1003216

    • PubMed
    • Google Scholar
21. 1. Goodall CP
    2. Bender RC
    3. Broderick EJ
    4. Bayne CJ

    (2004) Constitutive differences in cu/Zn superoxide dismutase mRNA levels and activity in hemocytes of Biomphalaria glabrata (Mollusca) that are either susceptible or resistant to Schistosoma mansoni (Trematoda)

    Molecular and Biochemical Parasitology 137:321–328.

    https://doi.org/10.1016/j.molbiopara.2004.06.011

    • PubMed
    • Google Scholar
22. 1. Gorbushin AM

    (2019) Derivatives of the lectin complement pathway in lophotrochozoa

    Developmental & Comparative Immunology 94:35–58.

    https://doi.org/10.1016/j.dci.2019.01.010

    • PubMed
    • Google Scholar
23. 1. Gordy MA
    2. Pila EA
    3. Hanington PC

    (2015) The role of fibrinogen-related proteins in the gastropod immune response

    Fish & Shellfish Immunology 46:39–49.

    https://doi.org/10.1016/j.fsi.2015.03.005

    • PubMed
    • Google Scholar
24. 1. Granath WO
    2. Yoshino TP

    (1984) Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

    Experimental Parasitology 58:188–193.

    https://doi.org/10.1016/0014-4894(84)90034-1

    • PubMed
    • Google Scholar
25. 1. Hahn UK
    2. Bender RC
    3. Bayne CJ

    (2000) Production of reactive oxygen species by hemocytes of Biomphalaria glabrata: carbohydrate-specific stimulation

    Developmental & Comparative Immunology 24:531–541.

    https://doi.org/10.1016/S0145-305X(00)00017-3

    • Google Scholar
26. 1. Hahn UK
    2. Bender RC
    3. Bayne CJ

    (2001) Killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata: role of reactive oxygen species

    The Journal of Parasitology 87:292–299.

    https://doi.org/10.1645/0022-3395(2001)087[0292:KOSMSB]2.0.CO;2

    • PubMed
    • Google Scholar
27. 1. Hambrook JR
    2. Kaboré AL
    3. Pila EA
    4. Hanington PC

    (2018) A metalloprotease produced by larval Schistosoma mansoni facilitates infection establishment and maintenance in the snail host by interfering with immune cell function

    PLOS Pathogens 14:e1007393.

    https://doi.org/10.1371/journal.ppat.1007393

    • PubMed
    • Google Scholar
28. 1. Hanington PC
    2. Forys MA
    3. Dragoo JW
    4. Zhang SM
    5. Adema CM
    6. Loker ES

    (2010) Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection

    PNAS 107:21087–21092.

    https://doi.org/10.1073/pnas.1011242107

    • PubMed
    • Google Scholar
29. 1. Hanington PC
    2. Forys MA
    3. Loker ES

    (2012) A somatically diversified defense factor, FREP3, is a determinant of snail resistance to schistosome infection

    PLOS Neglected Tropical Diseases 6:e1591.

    https://doi.org/10.1371/journal.pntd.0001591

    • PubMed
    • Google Scholar
30. 1. Hanington PC
    2. Zhang SM

    (2011) The primary role of fibrinogen-related proteins in invertebrates is defense, not coagulation

    Journal of Innate Immunity 3:17–27.

    https://doi.org/10.1159/000321882

    • PubMed
    • Google Scholar
31. 1. Hertel LA
    2. Adema CM
    3. Loker ES

    (2005) Differential expression of FREP genes in two strains of Biomphalaria glabrata following exposure to the digenetic Trematodes Schistosoma mansoni and Echinostoma paraensei

    Developmental & Comparative Immunology 29:295–303.

    https://doi.org/10.1016/j.dci.2004.08.003

    • PubMed
    • Google Scholar
32. 1. Howard SP
    2. Buckley JT

    (1985) Activation of the hole-forming toxin aerolysin by extracellular processing

    Journal of Bacteriology 163:336–340.

    https://doi.org/10.1128/JB.163.1.336-340.1985

    • Google Scholar
33. 1. Léonard PM
    2. Adema CM
    3. Zhang SM
    4. Loker ES

    (2001) Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata

    Gene 269:155–165.

    https://doi.org/10.1016/S0378-1119(01)00444-9

    • PubMed
    • Google Scholar
34. 1. Levashina EA
    2. Moita LF
    3. Blandin S
    4. Vriend G
    5. Lagueux M
    6. Kafatos FC

    (2001) Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, anopheles gambiae

    Cell 104:709–718.

    https://doi.org/10.1016/S0092-8674(01)00267-7

    • PubMed
    • Google Scholar
35. 1. Lie KJ
    2. Heyneman D
    3. Lim HK

    (1975) Studies on resistance in snails: specific resistance induced by irradiated miracidia of Echinostoma lindoense in Biomphalaria glabrata snails

    International Journal for Parasitology 5:627–631.

    https://doi.org/10.1016/0020-7519(75)90062-4

    • PubMed
    • Google Scholar
36. 1. Lie KJ
    2. Heyneman D

    (1975) Studies on resistance in snails: a specific tissue reaction to Echinostoma lindoense in Biomphalaria glabrata snails

    International Journal for Parasitology 5:621–625.

    https://doi.org/10.1016/0020-7519(75)90061-2

    • PubMed
    • Google Scholar
37. 1. Lockyer AE
    2. Spinks J
    3. Kane RA
    4. Hoffmann KF
    5. Fitzpatrick JM
    6. Rollinson D
    7. Noble LR
    8. Jones CS

    (2008) Biomphalaria glabrata transcriptome: cdna microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

    BMC Genomics 9:634.

    https://doi.org/10.1186/1471-2164-9-634

    • Google Scholar
38. 1. Lockyer AE
    2. Emery AM
    3. Kane RA
    4. Walker AJ
    5. Mayer CD
    6. Mitta G
    7. Coustau C
    8. Adema CM
    9. Hanelt B
    10. Rollinson D
    11. Noble LR
    12. Jones CS

    (2012) Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni

    PLOS ONE 7:e51102.

    https://doi.org/10.1371/journal.pone.0051102

    • PubMed
    • Google Scholar
39. 1. Loker ES
    2. Bayne CJ

    (1982) In vitro encounters between Schistosoma mansoni primary sporocysts and hemolymph components of susceptible and resistant strains of Biomphalaria glabrata

    The American Journal of Tropical Medicine and Hygiene 31:999–1005.

    https://doi.org/10.4269/ajtmh.1982.31.999

    • PubMed
    • Google Scholar
40. 1. MacKenzie CR
    2. Hirama T
    3. Buckley JT

    (1999) Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance

    Journal of Biological Chemistry 274:22604–22609.

    https://doi.org/10.1074/jbc.274.32.22604

    • PubMed
    • Google Scholar
41. 1. Moné Y
    2. Gourbal B
    3. Duval D
    4. Du Pasquier L
    5. Kieffer-Jaquinod S
    6. Mitta G

    (2010) A large repertoire of parasite epitopes matched by a large repertoire of host immune receptors in an invertebrate host/parasite model

    PLOS Neglected Tropical Diseases 4:e813.

    https://doi.org/10.1371/journal.pntd.0000813

    • PubMed
    • Google Scholar
42. 1. Mortality GBD
    2. GBD 2015 Mortality and Causes of Death Collaborators

    (2016) Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the global burden of disease study 2015

    Lancet 388:1459–1544.

    https://doi.org/10.1016/S0140-6736(16)31012-1

    • PubMed
    • Google Scholar
43. 1. Pila EA
    2. Gordy MA
    3. Phillips VK
    4. Kabore AL
    5. Rudko SP
    6. Hanington PC

    (2016a) Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host

    PNAS 113:5305–5310.

    https://doi.org/10.1073/pnas.1521239113

    • PubMed
    • Google Scholar
44. 1. Pila EA
    2. Tarrabain M
    3. Kabore AL
    4. Hanington PC

    (2016b) A novel Toll-Like receptor (TLR) Influences compatibility between the gastropod Biomphalaria glabrata, and the digenean trematode Schistosoma mansoni

    PLOS Pathogens 12:e1005513.

    https://doi.org/10.1371/journal.ppat.1005513

    • PubMed
    • Google Scholar
45. 1. Pila EA
    2. Li H
    3. Hambrook JR
    4. Wu X
    5. Hanington PC

    (2017a) Schistosomiasis from a snail's Perspective: Advances in Snail Immunity

    Trends in Parasitology 33:845–857.

    https://doi.org/10.1016/j.pt.2017.07.006

    • PubMed
    • Google Scholar
46. 1. Pila EA
    2. Peck SJ
    3. Hanington PC

    (2017b) The protein pheromone temptin is an attractant of the gastropod Biomphalaria glabrata

    Journal of Comparative Physiology A 203:855–866.

    https://doi.org/10.1007/s00359-017-1198-0

    • Google Scholar
47. 1. Portet A
    2. Galinier R
    3. Pinaud S
    4. Portela J
    5. Nowacki F
    6. Gourbal B
    7. Duval D

    (2018) BgTEP: an antiprotease involved in innate immune sensing in Biomphalaria glabrata

    Frontiers in Immunology 9:1206.

    https://doi.org/10.3389/fimmu.2018.01206

    • PubMed
    • Google Scholar
48. 1. Povelones M
    2. Bhagavatula L
    3. Yassine H
    4. Tan LA
    5. Upton LM
    6. Osta MA
    7. Christophides GK

    (2013) The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito anopheles gambiae

    PLOS Pathogens 9:e1003623.

    https://doi.org/10.1371/journal.ppat.1003623

    • PubMed
    • Google Scholar
49. 1. Richards CS

    (1975a) Genetic factors in susceptibility of Biomphalaria glabrata for different strains of Schistosoma mansoni

    Parasitology 70:231–241.

    https://doi.org/10.1017/s0031182000049696

    • PubMed
    • Google Scholar
50. 1. Richards CS

    (1975b) Genetic studies of pathologic conditions and susceptibility to infection in Biomphalaria glabrata

    Annals of the New York Academy of Sciences 266:394–410.

    https://doi.org/10.1111/j.1749-6632.1975.tb35118.x

    • PubMed
    • Google Scholar
51. 1. Sullivan JT
    2. Richards CS
    3. Joe LK
    4. Heyneman D

    (1982) Ribeiroia marini: irradiated miracidia and induction of acquired resistance in Biomphalaria glabrata

    Experimental Parasitology 53:17–25.

    https://doi.org/10.1016/0014-4894(82)90088-1

    • PubMed
    • Google Scholar
52. 1. Tsung A
    2. Klune JR
    3. Zhang X
    4. Jeyabalan G
    5. Cao Z
    6. Peng X
    7. Stolz DB
    8. Geller DA
    9. Rosengart MR
    10. Billiar TR

    (2007) HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling

    The Journal of Experimental Medicine 204:2913–2923.

    https://doi.org/10.1084/jem.20070247

    • PubMed
    • Google Scholar
53. 1. Vasquez RE
    2. Sullivan JT

    (2001) Further characterization of passively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata

    Journal of Parasitology 87:1360–1365.

    https://doi.org/10.1645/0022-3395(2001)087[1360:FCOPTR]2.0.CO;2

    • PubMed
    • Google Scholar
54. 1. Wilmsen HU
    2. Leonard KR
    3. Tichelaar W
    4. Buckley JT
    5. Pattus F

    (1992) The aerolysin membrane channel is formed by heptamerization of the monomer

    The EMBO Journal 11:2457–2463.

    https://doi.org/10.1002/j.1460-2075.1992.tb05310.x

    • PubMed
    • Google Scholar
55. 1. Wong SW
    2. Kwon MJ
    3. Choi AM
    4. Kim HP
    5. Nakahira K
    6. Hwang DH

    (2009) Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner

    Journal of Biological Chemistry 284:27384–27392.

    https://doi.org/10.1074/jbc.M109.044065

    • PubMed
    • Google Scholar
56. 1. Wu Y
    2. Li Q
    3. Chen XZ

    (2007) Detecting protein-protein interactions by far western blotting

    Nature Protocols 2:3278–3284.

    https://doi.org/10.1038/nprot.2007.459

    • PubMed
    • Google Scholar
57. 1. Wu XJ
    2. Dinguirard N
    3. Sabat G
    4. Lui HD
    5. Gonzalez L
    6. Gehring M
    7. Bickham-Wright U
    8. Yoshino TP

    (2017) Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni

    PLOS Pathogens 13:e1006081.

    https://doi.org/10.1371/journal.ppat.1006081

    • PubMed
    • Google Scholar
58. 1. Xu C
    2. Wang BC
    3. Yu Z
    4. Sun M

    (2014) Structural insights into Bacillus thuringiensis cry, cyt and parasporin toxins

    Toxins 6:2732–2770.

    https://doi.org/10.3390/toxins6092732

    • PubMed
    • Google Scholar
59. 1. Yassine H
    2. Osta MA

    (2010) Anopheles gambiae innate immunity

    Cellular Microbiology 12:1–9.

    https://doi.org/10.1111/j.1462-5822.2009.01388.x

    • PubMed
    • Google Scholar
60. 1. Yoshino TP
    2. Laursen JR

    (1995) Production of Schistosoma mansoni daughter Sporocysts from mother Sporocysts maintained in synxenic culture with Biomphalaria glabrata Embryonic (BGE) Cells

    The Journal of Parasitology 81:714–722.

    https://doi.org/10.2307/3283960

    • Google Scholar
61. 1. Zelck UE
    2. Becker W
    3. Bayne CJ

    (1995) The plasma proteins of Biomphalaria glabrata in the presence and absence of Schistosoma mansoni

    Developmental & Comparative Immunology 19:181–194.

    https://doi.org/10.1016/0145-305X(95)00012-I

    • PubMed
    • Google Scholar
62. 1. Zhang SM
    2. Léonard PM
    3. Adema CM
    4. Loker ES

    (2001) Parasite-responsive IgSF members in the snail Biomphalaria glabrata: characterization of novel genes with tandemly arranged IgSF domains and a fibrinogen domain

    Immunogenetics 53:684–694.

    https://doi.org/10.1007/s00251-001-0386-8

    • PubMed
    • Google Scholar
63. 1. Zhang SM
    2. Adema CM
    3. Kepler TB
    4. Loker ES

    (2004) Diversification of ig superfamily genes in an invertebrate

    Science 305:251–254.

    https://doi.org/10.1126/science.1088069

    • PubMed
    • Google Scholar
64. 1. Zhang SM
    2. Zeng Y
    3. Loker ES

    (2008) Expression profiling and binding properties of fibrinogen-related proteins (FREPs), plasma proteins from the schistosome snail host Biomphalaria glabrata

    Innate Immunity 14:175–189.

    https://doi.org/10.1177/1753425908093800

    • PubMed
    • Google Scholar

Author details

1. Hongyu Li

   1. Ocean College, Beibu Gulf University, Qinzhou, China
   2. School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Jacob R Hambrook

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Emmanuel A Pila

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Abdullah A Gharamah

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Jing Fang

   1. Ocean College, Beibu Gulf University, Qinzhou, China
   2. School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Xinzhong Wu

   1. Ocean College, Beibu Gulf University, Qinzhou, China
   2. College of Animal Sciences, Zhejiang University, Hangzhou, China

   Contribution

   Funding acquisition, Investigation, Methodology, Writing - review and editing

   For correspondence

   wuxzqinzhou@163.com

   Competing interests

   No competing interests declared

7. Patrick Hanington

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Project administration, Writing - review and editing

   For correspondence

   pch1@ualberta.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3964-5012

• 1,241

  views

• 216

  downloads

• 18

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.