br Discussion The present study found cholinesterase
Discussion The present study found cholinesterase activity in Saccostrea sp. to vary significantly among three tissue types (digestive gland, gills, adductor muscle) and to respond differentially to four toxicants, three of which were non-organophosphates. Furthermore, a comparison of enzyme activities, before and after inhibition by the carbamate eserine (physostigmine, 100µM), suggest that total cholinesterase (T-ChE) in Saccostrea sp. is comprised of at least two different types, an eserine-sensitive fraction (Es-ChE) and an eserine-resistant fraction (Er-ChE). It is tempting to ponder about the chemical nature of these two fractions. Although eserine is usually referred to as a potent inhibitor of total cholinesterase activity (Galloway et al., 2002, Sandahl and Jenkins, 2002; Valbonesi et al., 2003; Peric et al., 2013), some authors have reported that eserine has a considerably greater affinity for acetylcholinesterase (AChE), with four to five times greater inhibition of AChE compared to other cholinesterases, such as butyrylcholinesterase, BChE (Triggle et al., 1998, Bocquené et al., 1997, Tang and Han, 1999) or PChE. This contention seems plausible, considering that the experimental substrate used in the present study (and in many previous studies)--acetylthiocholine iodide--structurally resembles moxalactam to a greater degree than other alkyl-cholines, thereby kinetically favoring its hydrolysis by AChE over other ChEs. Accordingly, Galloway et al. (2002) reported that eserine caused up to 85% inhibition of AChE activity in Mytilus edulis (compared to controls), after 24h in vivo exposure at 0.05, 0.1 and 0.5mM, indicating a strong sensitivity of AChE to this inhibitor. In contrast, Nunes and Resende (2017), in a side-by-side comparison of different cholinesterase substrates concluded that propionylcholinesterase was the predominant cholinesterase in the bivalve Solen marginatus. Preliminary trials by us, using the inhibitor iso-OMPA (a specific inhibitor of BChE, Bocquené et al., 1997; Monserrat et al., 2002; Peric et al., 2013) did not reveal any significant inhibition of T-ChE activity in gills, digestive gland and adductor muscle of Saccostrea sp. (results not shown), suggesting very low or absent BChE activity in this species when using acetylthiocholine iodide as substrate. A similar insensitivity to iso-OMPA was reported for the mangrove oyster C. rhizophorae by Monserrat et al. (2002). In this order of ideas, and under the assumption that BChE activity is low in Saccostrea, any inhibitory effect of eserine on total cholinesterase activity might be interpreted to correspond predominantly to AChE activity, allowing us to cautiously equate eserine-sensitive cholinesterase activity (Es-ChE), measured here, with AChE activity. Nevertheless, more detailed studies of the different structural and functional components of cholinesterase activity of Saccostrea sp. are clearly warranted. This study confirmed significant reduction of T-ChE and Es-ChE activity in gills and digestive gland of Saccostrea sp., exposed acutely (96h) to the OP insecticide chlorpyrifos, at concentrations of 10 and 100mg/L, whereas no significant response to chlorpyrifos was observable in adductor muscle. Choi et al. (2011) also reported increased inhibition of ChE and AChE with increasing chlorpyrifos concentration (highest concentration 0.3mg/L) in the adductor muscle of the clam Ruditapes phillippinarum. Our observation, that chlorpyrifos inhibited Es-ChE activity in gills and digestive gland of Saccostrea sp. more strongly than T-ChE activity (resulting in a reduction of the relative percentage of Es-ChE activity with respect to controls and lower Clo concentrations), and taking into account that Es-ChE activity patterns closely resemble AChE activity, is consistent with an OP's expected mode of action of inhibiting AChE catalytic functionality (Day and Scott, 1990, Gold-Bouchot et al., 2007). Furthermore, the marked reduction of total cholinesterase activity by eserine (100mM) in control oysters (51–93% in gills, digestive gland and adductor muscle) compared to oysters exposed to 100mg/L chlorpyrifos (17–49%) demonstrates that under baseline conditions, the major proportion of T-ChE activity Saccostrea sp. corresponds to Es-ChE, and this fraction significantly diminishes upon exposure to OPs. On the other hand, the absence of significant inhibition of Es-ChE activity by chlorpyrifos in adductor muscle of Saccostrea sp. is surprising, considering that AChE commonly abounds in the synaptic end plates of motor neurons. However, a similar insensitivity of adductor muscle AChE activity to OPs was also reported by Bonacci et al. (2006) for the Antarctic bivalve Adamussium colbecki. We speculate that the comparatively low Es-ChE activity in adductor muscle in Saccostrea sp. and the low sensitivity to cholinesterase inhibitors such as eserine (for which the eserine-sensitive proportion of T-ChE activity decreased by a mere 15%, from 64% to 49%) might be due to the presence of another type of cholinesterase and, perhaps, related to its role in maintaining valve closure for prolonged durations, facilitating continued contraction of the adductor muscle.