Temperature and dissolved oxygen influence growth and digestive enzyme activities of yellowtail kingfish Seriola lalandi (Valenciennes, 1833)

A 5 week experiment was carried out with juvenile yellowtail kingfish Seriola lalandi to investigate the interactive effects of water temperature (21, 24, or 27°C) and dissolved oxygen regime (normoxic vs. hypoxic) on the growth rate, feed intake and digestive enzyme activity of this species. Specific growth rate (SGR) was highest at 24°C, regardless of oxygen regime, but the SGRs of the fish exposed to hypoxia at 21, 24 and 27°C were 13%, 20% and 17% lower, respectively, than the SGRs recorded for the fish reared under normoxic conditions. The digestive enzyme activities (i.e. trypsin, lipase and α‐amylase) were influenced by temperature but did not appear to be affected by dissolved oxygen concentration. Information about the effects of water temperature and dissolved oxygen on feeding, growth and digestive capacity of juvenile yellowtail kingfish could contribute to improving feed management decisions for production of this fish species under different environmental conditions.     

The responses of digestive enzymes in juvenile and adult Rapana venosa (Valenciennes, 1846) to different temperatures

The veined rapa whelk, Rapana venosa (Valenciennes, 1846), is an important and valuable fishery species; however, it has not been routinely artificially cultured on a large scale. Here, we studied the effect of temperature (6, 16, 22 and 28°C) on the digestive enzymes in R. venosa of different sizes (Juvenile (small, S: 1.2–1.8 cm) and adult (medium, M: 4.1–5.2 cm; large, L: 7.6–8.9 cm)) by detected the enzyme activity in different digestive glands (the salivary gland, Leiblein's gland (oesophageal gland) and liver) using spectrophotometric method, to determine the optimal temperature for artificial cultivation and to explore the feeding habits of this whelk. The optimal temperature was determined to be approximately 22°C. The food intake was significantly higher at 22°C than at other temperatures, and the activity of the digestive enzyme, trypsin, was at the highest in the main digestive gland of R. venosa, the liver, at 22°C. In addition, the observed presence of cellulase and α‐amylase may suggest that carbohydrates are necessary during R. venosa development. The results of this study will help in the formulation of technical procedures for R. venosa aquaculture and in preventing its overexploitation, and explained its invasion mechanism to some extent.     

Protease Activity and Partial Characterization of the Trypsin-Like Enzyme in the Digestive Tract of the Tropical Sierra Scomberomorus concolor

Abstract

Protease and trypsin-like activities of the digestive system of the tropical sierra Scomberomorus concolorwere evaluated. Sierra digestive tract extracts hydrolyzed specific substrates for trypsin, chymotiypsin, and leucine aminopeptidase At least eight bands of activity were observed by using SDS-PAGE. In PAGE and serineinhibition assays, one fraction resembled bovine trypsin. An ammonium sulfate (40-60%) trypsin-like fraction displayed different inhibitions with tosyllyschloromethyl ketone (96%) and soybean trypsin inhibitor (100%). Maximum activity was found using benzoyl-L-arginine-p-nitroanilide as substrate at pH 9.0 at 25°C and around 50°C at pH 7.8. The capacity of the trypsin-like fraction to work at different pHs (4, 7, and 9) at 25°C and different temperatures (0 and 40°C) at pH 7.8 were detected.     

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, <Emphasis Type="Italic">Hyriopsis</Emphasis> (<Emphasis Type="Italic">Hyriopsis</Emphasis>) <Emphasis Type="Italic">bialatus</Emphasis>

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.     

A comparative study of cellulase and xylanase activity in freshwater crayfish and marine prawns

Cellulase and xylanase digestive enzyme activities were compared in four freshwater crayfish (Genus Cherax) and three marine prawn (Genus Penaeus) species. Temperature and pH profiles for cellulase (endoglucanase) were found to be very similar in all species, with maximum activity occurring at 60°C and pH 5.0. Temperature and pH profiles for xylanase (endoxylanase) were also very similar in all crayfish species, with maximum activity occurring at 50°C and pH 5.0. Xylanase activity was not detected in the three prawn species examined. In addition, in vitro studies showed that most species were able to liberate glucose from carboxymethyl cellulose, indicating that cellulose substrates can be a source of energy for both crayfish and prawn species.     

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.     

Cross‐effects of dietary probiotic supplementation and rearing temperature on growth performance,digestive enzyme activities,cumulative mortality and innate immune response in seabass (Dicentrarchus labrax)

Probiotic influence on fish immune response and digestive capacity is extensively discussed in aquaculture. In this experiment, a feeding trial was carried out for 100 days to evaluate the cross‐effects of probiotic supplementation and rearing temperature (17, 20 and 23°C) in juvenile seabass (Dicentrarchus labrax). The experimental diet was supplemented with a commercial probiotic blend (Biomin AquaStar Growout) at 3 g/kg diet (5.23 × 10⁸ CFU/kg diet), and tested against a non‐supplemented diet (control). Growth performance and innate immune responses were analysed at 70 and 100 days of feeding, whereas digestive enzyme activities were determined at 100 days of feeding. At the end of the feeding trial, fish were subjected to a handling stress and cumulative mortality was recorded. Digestive enzyme activities were influenced by temperature, with α‐amylase and lipase activities peaking at the higher temperature (23°C) and trypsin at the lower temperature (17°C). Immune parameters showed a significant temperature versus feeding duration effect, with complement system (ACH50) and peroxidase peaking at 70 and 100 days of feeding, respectively. Poststress cumulative mortality was higher at the lowest temperature (17°C), especially in fish fed the control diet. In conclusion, water temperature was the main variable affecting the studied parameters, whereas the dietary probiotic supplementation had influence on the chymotrypsin activity and survival rate in seabass reared at 17°C.     

Partial characterization of alkaline proteases from viscera of vermiculated sailfin catfish Pterygoplichthys disjunctivus Weber, 1991

Vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991), a member of the Loricariidae family and an invasive species of several inland waters around the world, possess an enormous digestive tract representing about 10% of fish weight. Thus, the aim of this study was to partially characterize proteases from their digestive tracts. Azocasein digestion of the crude extract of intestine at different pH values and temperatures revealed the presence of alkaline proteases with optimum activities at pH 9.0 and 50°C. Incubation assays of the crude extract with inhibitors such as phenyl methyl sulfonyl fluoride, N-α-p-tosyl-l-lysine chloromethyl ketone, N-tosyl-phenyalanine chloromethyl ketone, benzamidine, pepstatin A and ethylenediamine tetra-acetic acid showed that trypsin and chymotrypsin are the main alkaline proteinases present. Zymography showed that the crude extract of Pterygoplichthys disjunctivus viscera contained proteases with molecular masses ranging from 21.5 to 116 kDa. Trypsin and chymotrypsin were inhibited by the following ions in decreasing order: Hg²⁺, Fe²⁺, Cu²⁺, Li⁺, Mg²⁺, K⁺, while Mn²⁺, and Ca²⁺ had no effect. Activities decreased continuously as the NaCl concentration increased from 0 to 30%. These results constitute important background information for future studies and for the potential biotechnological use of the crude digestive extract from this invasive species.     

Development of digestive biochemistry in the initial stages of three cultured Atherinopsids

The early development of the digestive biochemistry of three cultured Atherinopsids (two Odontesthes species and Chirostoma estor) was investigated. Relatively high lipolytic and high alkaline proteolytic activities were found in all species. In addition, gene expression of lipase and trypsin during the early development of C. estor not only indicates an early onset of the lipase activity but also demonstrates a major importance of protein digestion in this species. However, despite all of them share similar habitats, differences in their digestive activities were evident. The rise in the activities of brush border membrane enzymes correlated with the decrease in cytosolic activities, used as indicator of maturation of the digestive tract, was detected in both species of Odontesthes at 9 weeks after hatching. In contrast, no similar signs were measured in C. estor, this being in agreement to the long weaning period (nearly 5 months) reported for this species.     

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.     

Ontogeny of protease, amylase and lipase in the alimentary tract of hybrid Juvenile tilapia (Oreochromis niloticus × Oreochromis aureus)

Juvenile tilapias (Oreochromis niloticus × O. aureus), with average body weights of groups I, II and III of 55.14, 122.82, and 225.68 g respectively were used to study changes in the activity of protease, amylase, and lipase in different organs and sections of the alimentary tract. Additionally, tract containing digesta, which is closer to physiological conditions, may help reveal the mechanisms of whole chemical digestion process. Another group of tilapias (mean body weight of 111.81 ± 0.14 g) was used to analyze changes in activity of protease, lipase, and amylase in response to varying pH and temperatures. The activity of enzymes in all digestive organs increased when body weight increased from 55 to 122 g, and the activity further increased when the weights increased from 122 to 225 g. The rates of increase in digestive enzyme activity were significantly greater than the rates of growth of the fish. When the activity was compared in different sections of the tract, a similar regularity was found for the three enzymes in all fish. The highest amounts of activity were seen in the foregut and the lowest in the rectum, with the middle amount of activity in the hindgut. The pH ranges for the maximum activity of tilapia protease in the stomach and intestine were 1.6–3.3 and 9.5–10.5 respectively and the optimal temperature in these organs was 55°C. The pH ranges for the maximum amylase activity in the intestine and hepatopancreas were 6–7 and 7.5 respectively, and high amylase activity in these organs was found at 25–35°C. The activity of lipase in the intestine remained unchanged at pH 6.0–9.0 and with temperatures between 25 and 35°C. These results suggest that the ability of tilapia to utilize feed varies at different growth stages and remains relatively stable with changes in environmental pH and temperature, which should be taken into account when designing feed formulas for tilapia.     

Effect of diet composition on nutrient digestibility and digestive enzyme levels of gilthead sea bream (Sparus aurata L.)

An experiment was conducted to evaluate the effects of dietary composition on apparent digestibility coefficients of protein, fat and carbohydrate of gilthead sea bream. In addition α‐amylase and proteolytic activities were measured in the digestive tract of fish, held at 20±1°C. Six experimental diets were formulated containing approximately 40%, 45% and 50% protein, 11% and 21% fat and a starch level which fluctuated from 14% to 36%. Fish having an average weight of 100–130 g were used, fed at 1.5% of their body weight daily, and digestibility was measured using an indirect method. Enzyme activities were measured in the digestive tract of fish, fed diets containing a combination of 40%, 50% protein with 11%, 21% fat at 0.5, 5, 10, 24 h after a single meal. Starch digestibility was reduced with its level in the diet. It also negatively affected fat digestibility as well as protein digestibility, the last only at the high fat level. Dietary fat level had a negative effect on starch and protein digestibility. Fat affected also strongly α‐amylase levels in the digestive tract, while its effect on protease activity was of smaller magnitude. These results indicate that digestive enzyme activities and nutrient digestibility values are affected by dietary composition, carbohydrates and fat indicating the strongest effect.     

Effects of rearing temperature and dietary short‐chain fructooligosaccharides supplementation on allochthonous gut microbiota,digestive enzymes activities and intestine health of turbot (Scophthalmus maximus L.) juveniles

The gut microbiomes of fish play important roles in host development, digestion and health. Evidence suggests that abiotic factors, such as diet and rearing temperature, could affect fish gut microbiota. In this study, the effect of dietary short‐chain fructooligosaccharides (scFOS) on turbot intestinal health, microbiota and digestive enzymes was investigated at two rearing temperatures: 15 and 20 °C. Four practical diets were supplemented with scFOS at 0, 5, 10 and 20 g kg^?1. scFOS did not affect fish performance. PCR‐DGGE did not show differences in bacterial profiles between dietary treatments; however, the number of operational taxonomic units, richness and diversity were higher at 20 °C. Enzyme activities in the foregut were not affected by rearing temperature, while in the hindgut, enzyme activities were higher at 15 °C. Total alkaline protease, α‐amylase and lipase activities in the foregut were higher in fish fed 20 g kg^?1 scFOS. Prebiotic supplementation had no effect on hindgut α‐amylase activity, while lipase activity of fish reared at 20 °C was higher in fish fed diet with 5 g kg^?1 scFOS. No differences were observed in intestinal morphology. This was the first study to simultaneously evaluate the effect of dietary prebiotic level and rearing temperature on fish intestinal microbiota and health.     

Influence of high dietary α-tocopherol intakes on specific immune response, nonspecific resistance factors and disease resistance of healthy and aflatoxin B1-induced immunocompromised Indian major carp, Labeo rohita (Hamilton)

The effect of aflatoxin treatment and/or feeding of a high level of α‐tocopherol on immune response and disease resistance was investigated in Indian major carp, Labeo rohita. Group A served as a healthy control, group B was treated with aflatoxin, group C was fed a high level of α‐tocopherol whereas group D was exposed both to aflatoxin and a high level of dietary α‐tocopherol for 60 days. Aflatoxin B₁ (AFB₁) was injected once intraperitoneally into fish on the first day of the experiment (groups B & D). High levels of DL‐α‐tocopherol (1000 mg kg^–1 feed) were provided to healthy as well as AFB₁‐treated immunocompromised fish for 60 days (groups C & D). At the end of the experiment blood samples were assayed for changes in nonspecific immunity and humoral protein levels. Disease resistance against two common bacterial pathogens viz., Aeromonas hydrophila and Edwardsiella tarda were evaluated in all groups. Significant (P < 0.05) suppression of specific immunity as measured through haemagglutination (HA) titre against sheep red blood cells (SRBCs) as well as bacterial (formalin‐killed E. tarda) agglutination titre; nonspecific resistance factors viz., globulin level, serum bactericidal and lysozyme activities, neutrophil activities, and disease resistance against two bacterial pathogens only in aflatoxin‐treated fish with respect to the control group, clearly indicated the immunosuppressive nature of aflatoxin. Feeding of a high level of α‐tocopherol to AFB₁‐treated immunocompromised fish significantly (P < 0.05) raised specific immunity, nonspecific resistance factors and disease resistance capacity when compared with aflatoxin‐exposed fish. Disease resistance and enhancement of immune status through feeding of high levels of α‐tocopherol to healthy as well as AFB₁‐treated immunocompromised fish confirmed the potential of α‐tocopherol in carp feed for prevention of disease and for combating natural/environmental immunosuppressants.     

Fillet shelf-life of Atlantic halibut Hippoglossus hippoglossus L. fed elevated levels of α-tocopheryl acetate

Fish fillet quality may be influenced by the antioxidant level in preslaughter diet. Thus, the effects of dietary α‐tocopheryl acetate supplementation and feeding time on the flesh quality of farmed Atlantic halibut Hippoglossus hippoglossus L. were investigated. Halibut of mean initial weight of 312 ± 12.3 g were divided into two groups and fed commercial diets, supplemented with different levels of α‐tocopheryl acetate at the dietary inclusion levels of 189 and 613 mg kg^?1 diet. Fish were sampled after 6,9,12 and 24 weeks. Over the experimental period, they reached a final mean weight of 1320 ± 108.4 g. Tissue α‐tocopherol of fillet and liver was significantly affected by the levels of α‐tocopheryl acetate given with the diets (P < 0.001). In storage on ice, fillets of fish fed the diets high in α‐tocopheryl acetate exhibited significantly lower (P < 0.001) levels of lipid oxidation. The colour of fillets in all groups deteriorated slightly, but diet did not affect this process. Halibut fed the supplemented diets for longer periods were better protected against lipid oxidation (P < 0.001) and colour deterioration (P < 0.01) than those fed for shorter periods. However, after 9 days of storage, lipid oxidation levels were still extremely low [< 0.6 µg malondialdehyde (MDA) g^?1 fillet], even in fillets of fish fed the low α‐tocopheryl acetate diet for a short period preslaughter. Different slaughtering methods tested at the end of the trial showed that percussive stunning can delay the onset of rigor mortis by 8–12 h compared with bleeding of the fish. These results suggest that halibut fillets have enhanced shelf‐life stability even at low doses of dietary α‐tocopheryl acetate, and that other factors in the antioxidant defence mechanisms of the species might play a major role in the prevention of lipid oxidation.     

Morphogenese de l'appareil digestif et de la vessie gazeuse du turbot, Scophthalmus maximus L.Morphogenesis of the digestive system and swim bladder of the turbot, Scophthalmus maximus L.

The study is based on serial sections made on turbots raised at 18 ± 0.5°C from day 1 (post-hatching) to day 20. Histological changes of the digestive system and swim bladder were observed on a daily basis.At day 1, specimens (total length, Lt = 3.0–3.1 mm) present a digestive tract undifferentiated along its length and closed anteriorly. They belong to the embryonic period in spite of the presence of associated digestive glands (liver and pancreas).The larval period extends from day 2 to day 14–15 (Lt = 3.5–7.0 mm): the swim bladder differentiates and inflates, then the pneumatic duct binding the swim bladder to the stomach degenerates; regional differences appear along the digestive tract which starts to be functional.From day 15–16 (Lt = 6.9–7.7 mm) the juvenile period begins with the formation of gastric glands and the onset of gastric digestion. Two pyloric caeca are clearly visible and the number of intestinal folds and goblet cells increases progressively.     

Hot-water solubility of mantle collagens in several cephalopod molluscs

The mantle muscles of five cephalopod species, Todarodes pacificus, Photololigo edulis, Sepioteuthis lessoniana, Sepia esculenta, and Sepia longipes, were extracted with 0.1 M NaOH to prepare crude collagen fiber, called ‘residue after alkali extraction’ (RS-AL). Solubility of the collagens in water was examined in the temperature range 20–90°C. The collagens showed a similar tendency in solubility, which gradually increased depending on the treating temperature, and the values at 40–90°C were constantly less than 47.0% for all the species examined. In addition, the collagens were estimated to denature in the approximate temperature range of 37.5–42.5°C. These results suggest that the collagens in RS-AL from these species may have relatively high resistance to hot-water extraction even after their denaturation.     

Metabolic and digestive enzyme activity profiles of newly hatched spotted wolffish (Anarhichas minor Olafsen): effect of temperature

Three groups of newly hatched spotted wolffish (Anarhichas minor) were held at three different temperatures in order to determine relationships between metabolic, digestive and growth response in rapidly developing larvae. Growth rates were successfully modulated by temperature (5, 8 and 12 °C). Activity levels of trypsin and lactate dehydrogenase (LDH) were positively linked to specific growth rates at all temperatures. Trypsin showed a positive compensation (higher activity at lower temperature) whereas glycolytic enzymes (pyruvate kinase and Lactate dehydrogenase) and aspartate aminotransferase (AST) showed a negative compensation (lower activity at lower temperature). Citrate synthase was not affected by growth rate, indicating that the level of aerobic capacity was adequate in sustaining the high energetic needs associated with rapid growth early in the life of the spotted wolffish. In light of our results, it is suggested that protein digestion, as demonstrated by the activity profile of trypsin in relation to growth rate and temperature, is likely a key growth‐limiting agent during the early‐life stages of wolffishes. Our results are discussed in comparison with A. lupus, a closely related species displaying different temperature preferences and growth capacities.     

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

Digestive protease characterization, localization and adaptation in blacklip abalone (Haliotis rubra Leach)

Digestive protease in blacklip abalone (Haliotis rubra Leach) from whole gut extracts was found to have dual pH optima at pH 3 and 10 and an optimum temperature of 45 °C. Over the biologically relevant range of pH (5–8), protease activity dropped to a minimum at pH 5 (53% of the maximal rate at pH 3) rising gradually and continuously up to (and beyond) pH 8. Over the biologically relevant range of temperatures (9–24 °C), protease activity increased continuously with activity at 24 °C being 75% higher than activity at 9 °C. Protease digestion was relatively uniform in gut extracts from sections containing gut contents. Digestion thus appears to be significantly extracellular (in the lumen of the gut). Analysis of gut sections washed free of food matter suggests the anatomical origin of protease from activity of gut regions in the order: digestive gland » salivary gland ≈ stomach > crop > intestine > upper oesophagus. Whole gut protease levels did not alter in response to a high protein artificial diet.