In this work we show that two Schistosoma mansoni
glucose transporter (SGTP) genes, SGTP1 and SGTP4, are susceptible to suppression via RNAi. Of the two SGTPs targeted we find that SGTP4 is consistently better suppressed than SGTP1 using different siRNAs, long dsRNA and at two different life stages tested. This is consistent with the notion that genes expressed in schistosome tissues that are in direct contact with the environment (e.g. the tegument or the gut) are more efficiently suppressed by RNA interference compared to genes expressed in other tissues. SGTP4 is predominantly and perhaps exclusively expressed in the tegument 
whereas SGTP1 is additionally expressed in the internal tissues of the parasite, notably in the muscle 
. In the past we have noted that genes expressed predominantly or exclusively in the tegument (e.g. SmAQP) can be potently suppressed while those expressed both in the tegument and in internal tissues (e.g. SPRM1hc) are more poorly suppressed using the same protocols 
. This may reflect differences in the ability of dsRNAs to enter internal tissues or to the differential expression of RNAi pathway components in different organs.
The level of SGTP4 gene suppression in schistosomula is ~80%. This is the case when parasites are treated with dsRNA targeting SGTP4 alone or when treated with two siRNAs targeting SGTP4 and SGTP1. In a similar manner, the level of suppression of SGTP1 remains essentially the same when SGTP1 alone is targeted for suppression or when SGTP1 and SGTP4 are both targeted. These results support previous work 
showing that more than one gene can be suppressed at one time in schistosomes. Our quantitative data show that the RNAi machinery is not saturated by multiple siRNAs targeting different mRNAs.
The level of inhibition of glucose uptake into SGTP1-suppressed parasites is comparable to that seen for SGTP4-suppressed parasites. When SGTP1 alone is suppressed, glucose should still be able to enter the parasite tegument freely via the outer tegumental membrane transporter SGTP4. However, the movement of imported glucose further into the body of the SGTP1-suppressed parasites would then be impaired since this transporter is present on the tegumental basal membranes and on the membranes of other internal tissues. The inability of imported glucose to be efficiently transported out of the tegument and into the deeper tissues using SGTP1 would increase tegumental glucose concentrations and likely impede the further import of glucose by facilitated diffusion from the external environment. This is reflected in lower radiolabeled glucose being taken in to the SGTP1-suppressed parasites compared to controls.
When SGTP4 alone is suppressed, less glucose should enter the worms across the tegument compared to controls but any glucose that does enter and that is not utilized within the tegument should be efficiently transported inward via SGTP1. This would promote further glucose diffusion into the parasites via residual tegumental SGTP4 transporters. Parasites with both SGTP1 and SGTP4 genes suppressed exhibit a significantly greater impairment of radiolabeled glucose uptake compared with parasites that have had just one of the transporter genes suppressed. This likely reflects both a lower level of glucose uptake into the tegument via SGTP4 and an impaired ability to move that glucose into the internal tissues via SGTP1. Note that the level of glucose uptake in the doubly suppressed parasites is higher than that seen in parasites treated with a chemical inhibiter of facilitated glucose transporter protein function - cytochalasin B. This compound has been shown to block SGTP1 and SGTP4 function since it inhibits radiolabeled glucose uptake into Xenopus
oocytes that are expressing SGTP1 or SGTP4 
. The double SGTP knockdown parasites exhibited a higher glucose uptake (of ~30% versus untreated controls) compared to parasites treated with cytochalasin B (whose uptake was ~20% of untreated control parasites). Likely this reflects the high potency of cytochalasin B in almost completely shutting down all SGTP function. In contrast, RNAi leads to SGTP gene knockdown (but not gene knockout) and the presence of residual functional SGTP protein in the siRNA-treated groups does permit some label uptake. Residual protein includes any protein generated before siRNA administration as well as new protein derived from transcripts that survive the RNAi treatment. The diminished ability of SGTP-suppressed schistosomes to import glucose unequivocally demonstrates that these parasites do use both SGTP1 and SGTP4 to efficiently take in sugar. In a similar vein, earlier work reported that glucose uptake is impaired in schistosomes following exposure to SGTP antisense oligonucleotides 
. However, this work noted non-specific effects with some oligonucleotides and considerable variability between treatments, making the data equivocal 
. Previous work has demonstrated that SGTP1 is important for glucose uptake from the environment in the sporocyst life stage 
In order to establish whether the inability to import glucose by the SGTP-suppressed parasites had a detrimental impact on the worms, their viability was compared with that of control parasites in vitro and in vivo. Parasites in culture whose glucose transporter genes are suppressed show no significant phenotypic differences compared with controls, when they are maintained in medium with a high glucose concentration (10 mM) for up to 14 days. However, when these parasites are instead cultured in low glucose medium (0.05 mM) for 14 days, significantly fewer suppressed parasites survive compared with controls. This suggests that, in the sugar-poor environment, an impaired ability to import glucose upsets parasite metabolism and decreases viability. When SGTP-suppressed parasites infect mice, fewer of them survive to adulthood relative to controls. This is the case despite the fact that glucose concentrations in blood are high (~5 mM). These data suggest that the parasites' glucose demands in vivo are higher than in culture and this likely reflects the need for parasites in vivo to generate more energy (through glucose catabolism) to allow them migrate through tissues, invade the vasculature and combat host immune effectors.
The level of RNAi-mediated target gene suppression diminishes with time in culture. After 4 weeks in vitro
the level of suppression of SGTP1 is ~50% compared with ~65% at day 7 post treatment. For SGTP4 the suppression level at week 4 in culture is 70% compared with >95% at day 7. These data demonstrate that the RNAi effect remains substantial even after a month in culture. In contrast, equivalent parasites recovered from infected mice 4 weeks after RNAi treatment exhibit no remaining SGTP gene suppression. Those parasites that have survived in vivo
have SGTP mRNA levels at or even above control levels. Similar variable outcomes of RNAi in schistosomes ex vivo
compared to in vivo
have been reported in other studies 
. One hypothesis is that RNAi is variably effective in different parasites and/or that different individuals in the treated parasite population received different amounts of siRNA. Those in which SGTP knockdown is least effective, or that received less dsRNA, survive because the expression of their SGTP genes is minimally impaired. Another hypothesis is that worms in vivo
are more metabolically robust and this leads to a shorter half life of the dsRNA and/or its downstream effectors. In mammalian cells the longevity of the RNAi effect can depend on cell type: in non-dividing cells suppression can persist for several weeks whereas in rapidly dividing cells the effect may last only from 3 to 7 days. 
. Schistosomes in culture appear quiescent; they do not develop as quickly and fully as do parasites in infected animals and this may contribute to the persistence of gene suppression observed in the cultured worms.
In summary, this work shows that by demonstrably suppressing glucose transporter gene expression in schistosomes using RNAi, parasite feeding is hindered and this can significantly lower parasite viability. These findings provide direct evidence for the importance of SGTP1 and SGTP4 for schistosomes in importing exogenous glucose and show that the proteins are important for normal parasite development within the mammalian host.