The present study intended to characterize the predominant c

The present study intended to characterize the predominant cholinesterasic form found in tissues of three tropical fish species, namely Phalloceros harpagos Lucinda, 2008 (common name guaru), Pterygoplichthys pardalis Castelnau, 1855 (common name cascudo) and Astyanax altiparanae Garutti and Britski, 2000 (common name lambari). Guaru is representative of the neotropical am 580 being classified as a secondary consumer. P. harpagos belongs to the genus Phalloceros, which according to Machado et al. (2002), is characterized by small body size, ovoviviparous reproduction, with sexual dimorphism and larger and more abundant females. This species is omnivorous, demonstrating cannibal habits, however it feeds preferentially on aquatic and terrestrial insects, vegetables, algae and sediment. Lambari belongs to the genus Astyanax and has a very large species diversity; due to this fact, they are widely distributed in nearctic and neotropical environments (Ornelas-García et al., 2008). Females of A. altiparanae are larger than males. This fish species lives in shoals, which are organized by the size of each fish (Suzuki and Orsi, 2008). Lambari has the capacity of changing its feeding habits according to the habitat, being an omnivorous secondary consumer (Gomiero and Braga, 2003). Cascudo belongs to the genus Pterygoplichthys and is characterized by having large bony plaques and ventral mouth, are usually herbivorous and live in numerous populations (Page and Robins, 2006). P. pardalis is an algae consumer, has high fecundity, which results in a fast population growth, leading to adverse impacts on the wildlife from where this species is introduced (Hossain et al., 2018). All these fish species are endemic to South America, namely Brazil and Panamá (Page and Robins, 2006; Lucinda, 2008; Ornelas-García et al., 2008). In addition, the present study also intended to analyze and compare the sensitivity of main cholinesterases of each species, determined in the previous step, towards specific contaminants, namely the metals lead and copper (that have been implicated in anticholinesterasic effects; Cunha et al., 2007; De Lima et al., 2012; Nunes et al., 2014), and the anticholinesterasic pesticides carbaryl (reversible acetylcholinesterase inhibitor, widely used in agriculture and horticulture; Blacker et al., 2010; Hodgson, 2012) and dimethoate (irreversible acetylcholinesterase inhibitor, used as a neurotoxic insecticide; Gupta, 2016).
Materials and methods
Results
Discussion The main objective of this study was to characterize cholinesterase forms in two distinct tissues (brain and dorsal muscle) of three tropical fish species. A preliminary characterization of cholinesterasic isoenzymes is fundamental to allow their putative use in ecotoxicological assays, in order to correctly express enzymatic activities. The process of cholinesterases characterization is also important to allow understanding the potential impact that anticholinesterase agents may pose considering that distinct enzymatic forms may exhibit different sensitivity towards those agents (Nunes, 2011). It was also important to characterize the cholinesterase forms in different tissues of the three fish species, since their abundance may vary from tissue to tissue. The main cholinesterasic form present in brain and dorsal muscle of the species A. altiparanae showed a higher activity when incubated with ASCh as substrate; in addition, the cholinesterase activity in both tissues was inhibited by the three inhibitors used, but with distinctive profiles. An almost complete inhibition was obtained following incubation with extremely low levels of both eserine and BW284C51, suggesting the predominant presence of AChE in both tissues. Despite the occurrence of an inhibition with Iso-OMPA, this effect was not dose-dependent, and was only attained after incubating both tissues (brain and muscle) with the higher concentrations of this specific inhibitor. The almost complete inhibition at low doses of eserine and BW284C51, and the preference for ASCh as substrate, are evident signs that acetylcholinesterase was the major cholinesterase form in both tissues. However, the already reported significant inhibition by Iso-OMPA also occurred, albeit mild and not dose dependent; this effect was already described in previous studies, and has been related to the unspecific behavior of this inhibitor. At high concentrations, Iso-OMPA (which is the specific inhibitor of pseudo-cholinesterases) may also affect true cholinesterases and compromise their hydrolytic activities. This effect has been already reported for several other fish species, such as the marine fish Limanda limanda, Platichthys flesus and Serranus cabrilla, as described by Sturm et al. (1999) in a characterization study in brain and axial muscle. The authors described that in brain tissue of these three species, the predominant form was AChE, and that in the axial muscle, both forms AChE and BChE were present. Oreochromis niloticus brain has predominantly AChE, whereas its liver and muscle showed the presence of atypical ChE forms (Rodríguez-Fuentes and Gold-Bouchot, 2004). However, in the brain, liver and muscle tissues of Haemulon plumieri, AChE was the most abundant isoenzyme (Leticia and Gerardo, 2008). The fish species Pomatoschistus microps showed the presence of AChE as the predominant cholinesterasic form in brain homogenate (Monteiro et al., 2005). Similarly, the study conducted by Arufe et al. (2007) showed that the marine fish species Sparus aurata possesses a typical AChE form.