In vitro assay of plant protease inhibitors from four different sources on digestive proteases of rohu, Labeo rohita (Hamilton), fingerlings

The in vitro inhibitory effect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) fingerlings at two different pH ranges (8–8.5 and 10–11). That protein digestion occurs mainly in the alkaline condition in this fish without a stomach is evident from very high trypsin activity (0.95±0.04 benzoyl‐dl ‐arginine‐p‐nitroanilide U mg protein⁻¹) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 μg U⁻¹. More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 μg U⁻¹. Black gram at 11.0 μg U⁻¹ and soybean seed proteins at 62.75 μg U⁻¹ resulted in 50% and more than 30% inhibition of digestive protease activity in rohu fingerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in different dose–response curves for different protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned differences in inhibition. Each seed extract produced a characteristic profile of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely affect the digestive proteases in fish and hence there is a need to eliminate/reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.     

Results of spontaneous spawning of yellow snapper (Lutjanus argentiventris peters, 1869) reared in inland ponds in La Paz, Baja California Sur, México

Maturation and fecundity in two-year cycle of the yellow snapper (Lutjanus argentiventris) was studied as a potential fish-culture species in northwest Mexico. The results of this work will be used in future experiments to control sexual maturation by manipulation of photoperiod and temperature.     

In vitro protein digestibility of different grow‐out stages of spotted rose snapper (Lutjanus guttatus,Steindachner, 1869)

The aim of this study was to compare in vitro protein digestibility between two groups of fish, at early (21 g) and late stages (400 g) of spotted rose snapper Lutjanus guttatus, to evaluate the degree of hydrolysis (DH) and total amino acid release (TAAR) using crude extracts from stomach, pyloric caeca and intestine of 13 protein ingredients including marine, animal and plant meals. Degree of hydrolysis and TAAR were measured by a pH‐Stat method, and the PAGE‐Zymogram was also used as complementary technique. Differences in DH were found between both grow‐out stages mainly in the alkaline hydrolysis phase. Fish and squid meals (marine sources) had the highest DH and TAAR, followed by porcine meat and poultry meal by‐products from recycling sources, and soybean and canola meals (plant sources), which represent better protein sources for use in practical diets. Stomach zymograms showed two pepsin isoforms in both grow‐out stages. Pyloric caeca and intestine zymograms showed five bands with proteolytic activity in the early grow‐out stage, whereas four additional bands were found in late grow‐out stage. Alkaline proteases were identified as serine and metalloproteases. Thus, L. guttatus presents an ontogenetically differentiated digestive enzyme pattern that modifies the DH and TAAR of different protein sources.     

Proteolytic Activity in California Halibut Larvae (Paralichthys californicus)

The digestive tract of many marine fish larvae undergoes numerous morphological and functional changes during ontogeny that can substantially influence larval survival under culture conditions. Increasing our knowledge of the digestive capacity and nutritional requirements of the larvae of new candidate species for aquaculture will aid in the development of optimal feeding protocols and greatly improve production under hatchery conditions. In this study, we assess the proteolytic capacity of California halibut (Paralichthys californicus) larvae using biochemical and histological analyses. Newly hatched larvae were reared in a semiclosed recirculating system and fed with highly unsaturated fatty acid (HUFA)–enriched rotifers from hatching until 19 d posthatch (dph) and HUFA‐enriched Artemia nauplii thereafter. Total and specific activity of trypsin and leucine‐aminopeptidase (LAP) and acid and alkaline protease activities were assessed throughout development using spectrophotometric techniques. Trypsin‐like activity and LAP and alkaline protease activities were detected shortly after hatching and before the opening of the mouth. Acid protease activity was not detected until 36–40 dph, concomitant with the development of the gastric glands. The specific activity of trypsin and LAP showed two distinct peaks at 8 and 20 dph. The second peak coincided with the shift from rotifers to Artemia. Hence, newly hatched California halibut larvae possess alkaline proteolytic activity before first feeding. Based on the digestive capacity evaluated in this study and the timing of the development of the functional stomach, we propose that California halibut can be adequately weaned to formulated microdiets around 36 dph.     

Ontogenetic development of digestive enzymes in larval and juvenile crimson snapper Lutjanus erythopterus (Bloch 1790)

Ontogenetic development of digestive system in crimson snapper (Lutjanus erythopterus Bloch 1790) larvae was histologically and enzymatically (alkaline phosphatase, amylase, lipase, pepsin, trypsin) examined from hatching to 36 days post hatching (DPH). The ontogenetic development of crimson snapper larval fish ontogeny was divided into three distinct phases: phase I starting from hatch to the onset of exogenous feeding, phase II starting from first feeding (2–3 DPH) until the formation of gastric glands (13–14 DPH) and phase III beginning from the appearance of gastric glands and continuing onwards. The specific activities of amylase, lipase, trypsin showed sharp increase and reached to the maximum from hatch to 4 DPH, 10 DPH, 20 DPH, respectively, followed by a declining trend with irregular fluctuation. In contrast to other enzymes, the specific activities of alkaline phosphatase showed a gradual increase from hatch to 29 DPH, followed by a sharp increase towards 36 DPH. The specific activity of pepsin was firstly detected on 17 DPH and gradually increased towards the end of this study. The total activities of these five enzymes showed a gradual increase till 29 DPH, followed by a sharp increase towards 36 DPH except for amylase and lipase reaching maximum at 32 DPH. The present study provides better understanding of the digestive ontogeny of crimson snapper during the larval stage and a guide to feeding and weaning of this economically important fish in hatcheries.     

Organogenesis of the digestive system in Pacific red snapper (Lutjanus peru) larvae

This study reports the ontogenetic development of the digestive system of larval Pacific red snapper (Lutjanus peru), an important candidate species for aquaculture on the Pacific coast of Mexico. Histological sections of larvae were cut and dyed using the haematoxylin–eosin technique. The development of the digestive tract of Pacific red snapper larvae follows a general pattern of differentiation that can be divided into three stages. Stage I lasted from 1–3 days post hatching (DPH) and included the endogenous nutrition period; it was characterized by the initial differentiation of the digestive tract in preparation for the onset of exogenous feeding (3 DPH). At this time, the digestive tract was differentiated into buccopharynx, oesophagus, stomach anlage, anterior intestine, posterior intestine and a short rectum. The liver, pancreas and kidney were also present. The mouth and anus were open. Stage II occurred after first feeding, lasted for 16 days (4–23 DPH) and included both preflexion and flexion larvae. The main changes that occurred during this stage reflected the adaptation to exogenous feeding and the concomitant growth. Stage III (24–30 DPH) included post‐flexion larvae and started with the appearance of the gastric glands and pyloric caeca. The presence of the gastric glands suggests that early weaning during culture trials of the Pacific red snapper larvae may be possible at this early age.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

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.     

Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.     

Multidecadal otolith growth histories for red and gray snapper (Lutjanus spp.) in the northern Gulf of Mexico,USA

Dendrochronology (tree‐ring analysis) techniques were applied to develop chronologies from the annual growth‐increment widths of red snapper (Lutjanus campechanus) and gray snapper (Lutjanus griseus) otoliths sampled from the northern Gulf of Mexico, USA. Growth increment widths showed considerable synchrony within and across species, indicating that some component of environmental variability influenced growth. The final, exactly dated red snapper chronology continuously spanned 1975 through 2003, while the gray snapper chronology continuously spanned 1975 through 2006. To determine baseline climate‐growth relationships, chronologies were compared to monthly averages of sea surface temperatures, U winds (west to east), V winds (south to north), and Mississippi River discharge. The gray snapper chronology significantly (P < 0.01) correlated with winds and temperature in March and April, while the red snapper chronology correlated with winds in March. Principal components regression including springtime winds and temperature accounted for 28 and 52% of the variance in the red and gray snapper chronologies, respectively. These results indicate that snapper growth was favored by warm sea surface temperatures and onshore winds from the southeast to the northwest in March and April. Overall, this study provides preliminary, baseline information regarding the association between climate and growth for these commercially important snapper species.     

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles

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.     

Effect of dietary protein source and time on alkaline proteolytic activity of Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

A 4 × 3 factorial study was conducted to evaluate the effect of four experimental diets (a control diet and a 0, 50, and 100% fish meal replacement diet) and the period of time Nile tilapia, Oreochromis niloticus, were fed those diets (0, 20, and 40 days) on the alkaline proteolytic activity of the animals’ digestive tract, as well as their potential interaction. Significant differences (at P < 0.05) and a significant interaction were observed among dietary treatments for the alkaline proteolytic activity of tilapia after 40 days of feeding. This study confirmed that, under these experimental conditions, a 50% fish meal replacement formulation elicited the highest alkaline proteolytic activity in the digestive tract of tilapia, which resulted in the highest final weight and specific growth rate (SGR), but further research is needed to establish the relative contribution of the alkaline proteases to the overall proteolytic activity of this omnivorous fish species.     

Digestive enzyme activity during larval development of the Senegal sole (Solea senegalensis)

The activities of the main digestive enzymes (proteases, amylase, lipase) as well as those of acid and alkaline phosphatases were assessed during the larval development of the Senegal sole, Solea senegalensis. Important variations in specific activities of all the enzymes were observed during the period of study and were mostly related to the beginning or the end of metamorphosis. Both acid and alkaline protease activities were identified at early stages of development. Acid proteases accounted for only 10% of total protease activity in a 9 days-old larva, but exceeded 75% in a 33 days-old individual. Maximum lipase activity was related to the development of exocrine pancreas (days 6 to 10) and to metamorphosis. It is suggested this can be related to the metabolism of lipid reserves taking place during this morphological change. Activity of alkaline phosphatase decreased from day 5 to day 20 but therafter revealed a sharp increase, possibly linked to the development of enterocytes. The pattern of development of the main digestive enzymes found in S. senegalensis is similar to that described in other flatfish species.     

Digestive enzyme activity during early larval development of the Cuban gar Atractosteus tristoechus

The ontogenesis of digestive enzymes (proteases, amylases, lipases, and phosphatases) in Cuban gar Atractosteus tristoechus was determined in larvae between 5 and 18 days after hatching (DAH). Variations in specific activities of most enzymes were related to the transition from endogenous to exogenous feeding and to the transition from the larval to the juvenile stage. Alkaline protease activity was not detected until 8 DAH in contrast to acid protease activity, which was quantifiable at 5 DAH. Acid protease activity was consistently higher than alkaline protease activity, indicating the presence of a functional stomach in the early stages of larval development. The acid protease activities of larvae and adults were compared by means of zymogram analysis. Four acid protease bands were found in adults (two more than in larvae). This result is the first time that more than one band of acid proteolytic activity has been found in Lepisosteidae. High lipase activity indicated the importance of lipid utilization, particularly during yolk-sac absorption. In contrast to the other enzymes studied, amylase activity was consistently low, probably due to the strictly carnivorous diet of gar larvae and their low capacity for carbohydrate digestion. High activities of aminopeptidase and acid and alkaline phosphatases suggest intestinal absorption. This result, together with the existence of a short gut and a lower proteolytic activity in the intestine than in the stomach, suggest that most of the proteolytic activity takes place in the stomach, while the primary function of the intestine is nutrient uptake.     

Characterization of protease activity in developing discus Symphysodon aequifasciata larva

The activity of different protease classes was monitored in developing discus (Symphysodon spp.) larvae using a combination of biochemical assays and substrate SDS–PAGE techniques. Results showed the presence of alkaline proteases of serine proteases such as trypsin with a significant increase in activity levels detected beginning 3 days after hatching. Other alkaline proteases such as metallo‐proteases and chymotrypsin, a type of serine protease, were only detected in older larval stages, at around 20–30 days after hatching. Acidic protease activity was very low during the first 20–25 days of development before showing a significant (P < 0.01) rise. This is despite the formation of a stomach observed 10 days after hatching. Based on the development of the protein digestive system observed, the use of microdiets to replace Artemia should be considered 25 days after hatching.     

Phytic Acid‐Induced Inhibition of Digestive Protease and α‐Amylase in Three Indian Major Carps: An in vitro Study

Antinutritional effects of phytic acid (myoinositol hexaphosphate, IP6) on growth and digestibility in fish have been reported. However, specific effect of IP6 on the digestive enzymes in fish has not been addressed. In this study, inhibitory effect of synthetic IP6 (Phytic acid sodium salt, 90% purity) on the activity of the digestive protease and α‐amylase in rohu, Labeo rohita; catla, Catla catla; and mrigal, Cirrhinus mrigala has been investigated in vitro. Graded levels (12.5, 25, 50, 100, 150, and 200 µg/mL) of IP6 were added to the reaction mixtures containing enzyme extracts and substrate solution in triplicate to detect any change in enzyme activity. Results of the experiment revealed that IP6 significantly inhibit/lower activities of the digestive enzymes in a dose‐dependent manner, as evident from the regression equations (F values significant at P < 0.001 level). Apparently, irrespective of the fish species studied IP6‐induced inhibition of α‐amylase activity was greater than protease activity. Among the three fish species studied, C. mrigala appeared to be more sensitive to IP6 for both α‐amylase and protease activity. Enzyme activity was least affected in C. catla. Results of the study might raise concern while incorporating IP6 rich plant‐derived feed ingredients in aqua feed preparation.     

Digestive dynamics during chyme formation of Octopus maya (Mollusca,Cephalopoda)

In the present study, digestive dynamics, measured through the changes in the in situ pH along the digestive tract, was evaluated along with the glycogen concentration in the digestive gland (DG) and the proteolytic enzyme activity of the gastric juice (GJ) (both acid and alkaline proteases) to obtain useful information that will allow for the understanding of the DG's function and the role that it plays as an extracellular digestive enzyme source and for energy storage, providing new information on the digestive physiology of Octopus maya. The results showed that pH along the digestive tract changed according to the postprandial time following the food transit. At the beginning, the pH on crop (St) was 5, changing to 6 when the food arrived. Similar changes were observed on the caecum (Ce) and DG. Glycogen from DG is used as a source of energy during digestion and recovered 8 h after feeding. Maximum activities of digestive gland GJ enzymes were observed 6 h after feeding, indicating that chyme enzymes are still active when they arrive at the DG's lumen. The presence of food in the digestive tract modifies the pH, which, in combination with GJ, favours the activity of the released enzymes of O. maya.     

Inhibition of protease activities in discus Symphysodon spp. by three plant meals

Based on biochemical assays and electrophoretical methods, the inhibitory effects of three plant meals (soybean meal, wheat meal, winged bean meal) on digestive alkaline proteases of discus were investigated. Casein assays revealed that increasing levels of soybean meal caused a linear inhibitory effect on activity of protease. SDS-PAGE images revealed that trypsin and chymotrypsin were the inhibited enzymes. Soybean showed the lowest inhibition rate followed by wheat meal and raw winged bean. There was a quadratic relationship between wheat meal levels and its inhibition of protease acitivity. The highest inhibitory effect was obtained with the winged bean meal with inhibition of caseinolytic activities ranging from 3.6–98.6%. Results from this study showed the potential of both soybean meal and wheat meal as ingredients for practical diet for discus, while demonstrating the need for further improvement in processing method for winged bean meal.     

Development of digestive enzyme activity in spotted rose snapper,Lutjanus guttatus (Steindachner, 1869) larvae

We describe digestive enzyme activity during the larval development of spotted rose snapper, Lutjanus guttatus. Trypsin, chymotrypsin, leucine aminopeptidase, pepsin, amylase, lipase, and acid and alkaline phosphatase activities were evaluated using spectrophotometric techniques from hatching through 30 days. The spotted rose snapper larvae present the same pattern of digestive enzyme activity previously reported for other species in which pancreatic (i.e., trypsin, chymotrypsin, amylase, and lipase) and intestinal (i.e., acid and alkaline phosphatases and leucine aminopeptidase) enzymatic activities are present from hatching allowing the larvae to digest and absorb nutrients in the yolk-sac and live prey by the time of first feeding. The digestive and absorption capacity of the spotted rose snapper increases during the larval development. A significant increase in individual activity of all enzymes occurs at 20 DAH, and around 25 DAH, the juvenile-type of digestion is observed with the appearance of pepsin secreted by the stomach, suggesting that maturation of the digestive function occurs around 20–25 DAH. Our results are in agreement with a previous suggestion that early weaning may be possible from 20 DAH. However, the patterns of enzymatic activities reported in our study should be considered during the formulation of an artificial diet for early weaning of the spotted rose snapper.     

Differences in the digestive biochemistry between the intertidal clam, Ruditapes decussatus, and the subtidal clam, Venerupis pullastra

The present study was aimed to evaluate differences in physicochemical characteristics (pH and temperature optima) of the main digestive enzymes (amylase, cellulase, laminarinase, and protease) in two commercially important bivalves, the intertidal clam, Ruditapes decussatus, and the subtidal clam, Venerupis pullastra, as well to assess individual variability of such activities. The assays were performed on extracts obtained from the digestive glands and crystalline styles of individuals collected in the wild and laboratory-conditioned during 1 week. In both species, maximum activity for amylase and cellulase was measured at pH 6 and that of laminarinase at pH 5.5, while pH profiles of protease activity showed important differences between them. Temperature profiles also showed interspecific variations, mainly in laminarinase. Carbohydrase activities were comparatively much higher in R. decussatus and proteolytic activities in V. pullastra. The observed differences both in physicochemical characteristics and relative importance of digestive enzymes are discussed in relation to the amount and type of food available in their respective habitats.     

Partial characterization of digestive proteases of the three‐spot cichlid Cichlasoma trimaculatum (Günter 1867)

Partial characterization of digestive proteases in the three‐spot cichlid Cichlasoma trimaculatum juveniles was conducted. It was determined that there is higher alkaline proteases activity (3.95 ± 0.32 IU mg^?1 protein) compared to acidic proteases (2.01 ± 0.57 IU mg^?1 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^?1 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.