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Immune factors in snails of the genus Biomphalaria are critical for combating Schistosoma mansoni, the predominant cause of human intestinal schistosomiasis. Independently, many of these factors play an important role in, but do not fully define, the compatibility between the model snail B. glabrata, and S. mansoni. Here, we demonstrate association between four previously characterized humoral immune molecules; BgFREP3, BgTEP1, BgFREP2 and Biomphalysin. We also identify unique immune determinants in the plasma of S. mansoni-resistant B. glabrata that associate with the incompatible phenotype. These factors coordinate to initiate haemocyte-mediated destruction of S. mansoni sporocysts via production of reactive oxygen species. The inclusion of BgFREP2 in a BgFREP3-initiated complex that also includes BgTEP1 almost completely explains resistance to S. mansoni in this model. Our study unifies many independent lines of investigation to provide a more comprehensive understanding of the snail immune system in the context of infection by this important human parasite.

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13Immune factors in snails of the genus Biomphalaria are critical for combating 14 Schistosoma mansoni, the predominant cause of human intestinal schistosomiasis. 15 Independently, many of these factors are known to play an important role in, but not fully 16 define, the compatibility between the model snail B. glabrata, and S. mansoni. Here, we 17 demonstrate association between four, previously characterized humoral immune 18 molecules; BgFREP3, BgTEP1, BgFREP2 and Biomphalysin. We also identify unique 19 immune determinants in the plasma of S. mansoni-resistant B. glabrata that explain the 20 incompatible phenotype. These factors coordinate to initiate haemocyte-mediated 21 deestruction of S. mansoni sporocysts via production of reactive oxygen species. The 22 inclusion of BgFREP2 in a BgFREP3-initiated complex that also includes BgTEP1 almost 23 completely explains resistance to S. mansoni in this model. Our study unifies many 24 109 that BgFREP3 binds to S. mansoni sporocysts without any other soluble plasma factors, 110 yet BgFREP2 relies on BgTEP1. However, BgFREP3 still interacts with BgTEP1 to form 111 unique immune complexes, which significantly enhance the ability of haemocytes and 112 6 plasma from S. mansoni-susceptible B. glabrata (M-line) to kill S. mansoni sporocysts. A 113 more striking finding is that the combination of BgFREP3, BgTEP1 and BgFREP2 114 renders M-line haemocytes capable of killing S. mansoni sporocysts at nearly the same 115 level as S. mansoni-resistant BS-90 haemocytes. Reactive oxygen species (ROS) is 116 shown to play an important role during this haemocyte-mediated killing of S. mansoni 117 sporocysts. These results provide insight into how the numerous previously characterized 118 immune factors known to be important in the anti-S. mansoni immune response to B. 119 glabrata are acting in concert to defend the snail host.120 Results 121BgFREP3 interacts with BgTEP1, Biomphalysin, BgFREP2 and other BgFREP3 122 variants in snail plasma. 123 To identify the factors in snail plasma which interact with BgFREP3, we produced 124 recombinant BgFREP3 (rBgFREP3, GenBank: AAK28656.1) using an insect expression 125 system ( Fig. 1A and Figure 1 -figure supplement 1) and performed a series of pull-down 126 experiments. We initiated our investigations with BgFREP3 because we observed that it 127 is expressed at greater abundance in BS-90 compared with M-line snails. This 128 observation was confirmed using Western-blot that showed that the hemolymph of BS-90 129 snails contain more BgFREP3 than does M-line snail hemolymph (Fig. 1B). Among the 130 proteins identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) 131 in the eluent of these pull-down experiments was BgTEP1 (Fig. 1C). We performed four 132 rBgFREP3 pull-down experiments, each yielded a band at approximately 200-kDa, which 133 corresponded to the full-length BgTEP1, which was identified from both M-line and BS-134 790 strains (Fig. 1C). In three instances, we cut the bands and sent for mass spectromet...