Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

Summary The ontogeny of the digestive tract in Cichlasoma urophthalmus was studied by means of optical microscopy from hatching to 30 days post‐hatching (dph; 855 degree days, dd). The development of the digestive system in this precocial species was a very intense and asynchronous process, which proceeded from both distal ends interiorly. At hatching, the digestive tract consisted of a straight tube with a smooth lumen dorsally attached to the yolk‐sac. The digestive accessory glands were already differentiated and eosinophilic zymogen granules were visible in the exocrine pancreas. At the onset of exogenous feeding between 5 and 6 dph (142.5–171.0 cumulative thermal units, CTU), the buccopharynx, oesophagus, intestine, liver and pancreas were almost completely differentiated, with the exception of the gastric stomach that completed its differentiation between 11 and 14 dph (313.5–399.0 CTU). The development of gastric glands at 14 dph and the differentiation of the stomach in the fundic, cardiac and pyloric regions at 19 dph (541.5 CTU) were the last major events in digestive tract development and designated the onset of the juvenile period. Remnants of yolk were still detected until 16 dph (456.0 CTU), indicating a long period of mixed nutrition that lasted between 10 and 11 days (285.0–313.5 CTU). The results of the organogenesis of larvae complement previous data on the functionality of the digestive system and represent a useful tool for establishing the functional systemic capabilities and physiological requirements of larvae to ensure optimal welfare and growth under aquaculture conditions, which might be useful for improving current larval rearing practices for this cichlid species.

Five experimental diets with different carbohydrates/lipid ratio (CHO:L, 0.75, 1.28, 2.10, 2.52, 4.63) were formulated. Fish were randomly assigned by triplicate (10 fish per tank, 0.50 ± 0.01 g). Growth was registered every 15 days until end of the experiment (45 days). Samples were taken for analysis of chemical carcass composition, blood chemistry, glycogen and lipid liver content, digestive and metabolic enzyme activities. Results showed that survival, growth performance parameters and plasma glucose were not affected by treatments (p > 0.05). Lipids of carcass and liver, as well as triglycerides and plasma cholesterol increase significantly as CHO:L ratio decreased. While an inversely proportional tendency was observed for carcass protein and liver glycogen (p < 0.05). Digestive enzymes did not show significant differences among treatments (p > 0.05). Finally, Hexokinase (HK), glucokinase (GK), phosphoenol pyruvate carboxykinase (PEPCK) and fructose‐1,6‐biphosphase (FBPase) showed high regulation by carbohydrates up to the CHO:L ratio of 2.10 (p < 0.05), while pyruvate kinase (PK) activity was not significantly affected by the CHO:L ratio. By the other side, glucose‐6‐phosphate dehydrogenase (G6PD) tends to significantly increase as the CHO:L ratio increases. We conclude that A. tropicus fry have a high capacity to utilize carbohydrates substituting lipids as energy source in balanced diets.

Partial characterization of digestive proteases in the threespot cichlid Cichlasoma trimaculatum juveniles was conducted. It was determined that there is higher alkaline proteases activity (3.95 AE 0.32 IU mg À1 protein) compared to acidic proteases (2.01 AE 0.57 IU mg À1 protein). Optimal temperature for alkaline proteases is 60°C which resulted in more thermostability to temperature changes. On the other hand, optimal temperature for acidic proteases is 50°C. Optimal pH for acidic proteases was pH 2, while for alkaline proteases, it was pH 10, which resulted in more stability in relation to pH changes than acidic protease. The use of specific inhibitors and the SDS-PAGE electrophoresis analysis revealed seven types of bands for alkaline proteases, which make evident the main presence of serine proteases. In acidic proteases, more than 98 g kg À1 of the activity was inhibited with pepstatin A inhibitor. Therefore, it is evident that C. trimaculatum digestion is composed by acidic and alkaline proteases; thus, it should be considered an omnivorous fish.