The effect of ESPs on haemocyte NO production is strain specific; haemocytes from schistosome-resistant snails displayed significantly increased NO output after ESP challenge. Previous studies using the same snail strains have highlighted S. mansoni ESPs' ability to attenuate ERK signalling in haemocytes from the susceptible B. glabrata strain, with no effect in those from the resistant strain . In the present study, inhibition of ERK signalling in haemocytes from both snail strains led to down-regulation of NO production, demonstrating that NO production is partly under the control of ERK signalling, which is consistent with earlier work on L. stagnalis haemocytes .
A highly sensitive NO probe (DAF-FM diacetate) which detects intracellular NO was used in this investigation. This probe has been used previously in other invertebrate models including: the squid, Euprymna scolopes, the moth, Manduca sexta, the sea squirt, Ciona intestinalis and the snail L. stagnalis [5, 19–21]. In molluscs, the production of NO has also been studied in the nervous system [10, 11]. Franchini et al.,  identified an immunoreactive NOS-like protein in molluscan haemocytes that could be induced by stimulating the cells with E. coli, while haemocytes from the bivalve M. galloprovincialis were shown to generate superoxide and nitrites following exposure to phorbol myristate acetate (PMA), laminarin and during yeast cell phagocytosis . Lipopolysaccharide (LPS) does not induce NO generation in L. stagnalis or M. galloprovincialis haemocytes [5, 6], but does stimulate an NO response in the clam Ruditapes decussates .
Oxygen-dependent killing mechanisms in molluscs such as B. glabrata are known to play a crucial role in killing schistosome sporocysts [7, 25, 26]. It has been hypothesised previously that differences exist between schistosome-resistant and schistosome-susceptible B. glabrata strains in their oxygen-dependent killing mechanisms . Schistosoma mansoni ESPs are known to affect the physiology of haemocytes; cell motility, phagocytosis of zymosan particles, and the production of reactive oxygen metabolites are differentially modulated in susceptible and resistant B. glabrata strains by S. mansoni ESPs [28–30].
In the present study, NO levels in B. glabrata haemocytes from susceptible and resistant snail strains were found to be affected differentially by S. mansoni ESPs. Haemocytes from resistant snails had a significantly greater increase (3.3 times) in NO production than controls following 5 h ESP challenge, while haemocytes from susceptible snails were not significantly affected. Furthermore, basal levels of NO (from unchallenged haemocytes) were significantly different between the two snail strains, with haemocytes from susceptible snails producing relatively more NO over time. The reason for the basal NOS activity in extracted haemocytes is unknown; similar basal activities were also observed previously in extracted L. stagnalis haemocytes . Earlier studies have shown that haemocytes from schistosome-resistant B. glabrata strains maintain higher levels of intracellular superoxide and hydrogen peroxide when stimulated with S. mansoni ESPs and PMA, respectively, compared to susceptible strains [28, 31]. Hahn et al.  reported no differences in the relative production of reactive oxygen species (ROS) in haemocytes from susceptible and resistant snail strains following stimulation with carbohydrates known to be present on the schistosome surface, while Humphries and Yoshino  reported no effect of S. mansoni ESPs on hydrogen peroxide generation in haemocytes from resistant B. glabrata.
Human recombinant interleukin-2 (IL-2), a known NO stimulant for mammalian macrophages, was found to enhance considerably NO production in M. galloprovincialis haemocytes by approximately 13-fold, an effect which was reduced in the presence of a protein kinase A (PKA) inhibitor . Thus a cAMP-dependent protein kinase might be involved in NO generation in molluscs, together with PKC, which was found to be an important NO regulator in L. stagnalis haemocytes . Here, in the presence or absence of ESPs, the MEK inhibitor, U0126, significantly reduced NO production in susceptible and resistant B. glabrata haemocytes. The inhibitor also substantially attenuated ERK phosphorylation in haemocytes from both snail strains. This implies a role for ERK signalling in NO output through NOS regulation, similar to that reported in L. stagnalis haemocytes . Extracellular hydrogen peroxide generated by stimulating B. glabrata haemocytes with PMA, galactose-conjugated BSA, or through the process of encapsulation and phagocytosis is also partially regulated by ERK signalling [32, 33].
Earlier work has shown that haemocytes from schistosome-susceptible B. glabrata challenged with ESPs display significantly reduced ERK phosphorylation . In the current investigation, haemocytes from the same snail strain had significantly reduced NO levels following exposure to the ERK inhibitor, U0126. However, reduced NO output in the presence of U0126 could also be attributed to an effect of U0126 on an ERK-like protein, not recognised by the anti-phospho ERK antibody used here and in the study by Zahoor et al. ; only one ERK-like protein was detected by this antibody in B. glabrata haemocytes extracts, whereas two ERK isoforms are sometimes detected in L. stagnalis . Moreover, the increased NO output observed here in resistant snail haemocytes following ESP exposure might be a consequence of the sustained ERK phosphorylation previously seen in these cells under ESP challenge . In addition, ESPs may be influencing the activities of other cell signalling pathways, such as protein kinase C (PKC) or PKA, resulting in modulation of intracellular NO production.
Given the cytotoxic effects of NO, longer-term effects of ESPs on NO production in haemocytes from both snail strains may influence survival of invading schistosomes in vivo. In the present in vitro study, schistosome-susceptible snail haemocytes have been shown to produce more intracellular NO under basal conditions than those of resistant snails (even though resistant snails have been reported to possess twice as many haemocytes ). Why this phenomenon exists is currently unknown, but may involve the haemocytes maintaining different intracellular NO equilibriums. Intracellular ROS equilibriums are known to play an important role in allowing long term survival of the host and the parasite; in the case of malaria, increased oxidative stress in the host's erythrocytes can lead to exacerbated disease progression, while partial inhibition of macrophage NO production by Toxoplasma gondii enhances the parasite's survival [34, 35]. Furthermore, an intracellular NO equilibrium may be physiologically important; NO is involved in a number of cell signalling events and, depending on the cellular environment, can promote either cell survival or cell death [36, 37].