Biosynthesis of Astacus protease, a digestive enzyme from crayfish

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

The ultrastructure of the sinus gland of the crayfish Astacus leptodactylus (Nordmann)

Summary An ultrastructural study of the sinus gland of the crayfish Astacus leptodactylus demonstrates that this gland is mainly composed of glial cells, axons and axon terminals. On the basis of the size, shape and electron density of the neurosecretory granules, we could distinguish five different types of axon terminals.     

Effect of different diets on digestive enzyme activities,in vitro digestibility,and midgut gland structure in juvenile crayfish,Cherax quadricarinatus

This study investigated the effects of food quality on digestive enzyme activities, in vitro protein digestibility and histological traits of the midgut gland in juvenile crayfish Cherax quadricarinatus. Animals of a wide weight range were fed different diets: two commercial diets with high or low lipid content (high lipid and low lipid, respectively) and were compared with a reference diet (RF) previously formulated for this species. Proteinase, lipase and amylase activities were significantly influenced by diet and weight. Specific trypsin activity was significantly higher for crayfish fed with the HL diet. Trypsin activity depended on diet and weight. Protein digestibility showed that HL was the most digestible diet and RF the least. The weight of the animals did not affect protein digestibility. Structural disorganization, hypertrophy of B‐cells and presence of large vacuoles in R‐cells were mainly observed in juveniles fed with HL, indicative of malnutrition. Thus, our data suggest that the HL diet would not be the most appropriate for C. quadricarinatus, while RF diet would be more convenient for culture of this species.     

APPLIED ISSUES Replacement of the native crayfish Astacus astacus by the introduced species Pacifastacus leniusculus in a Swedish lake: possible causes and mechanisms

1. The introduced North American crayfish Pacifastacits leniusculus is currently replacing the native crayfish Astacus astacus in a Swedish lake. Using field data from co-occurring populations on life-history traits and population size structure of the two species, this study evaluated possible mechanisms behind the replacement. 2. Pacifastacus showed a higher capacity for population increase; the species had a higher individual growth rate, reached sexual maturity at a smaller size and lower age, and had a higher per capita egg production than Astacus. 3. A higher frequency of non-lethal injuries in Astacus suggested the predominance of Pacifastacus in interference interactions. These results support a competitive exclusion hypothesis. 4. A dramatic change in relative abundance of the two species in recent years was the result of an almost complete cessation in recruitment of young-of-the-year (YOY) in Astacus. Poor recruitment in Astacus was probably caused by the combined effects of interspecific competition and predation, resulting in increased mortality among YOY Astacus, and by reproductive interference, suppressing the less common species. 5. The results support the hypothesis that the observed replacement of Astacus by Pacifastacus is governed by a combination of several interacting mechanisms, of which     

The effect of adipokinetic hormones on the activity of digestive enzymes

The role of the adipokinetic hormone (AKH) in the control of protease, amylase and lipase activities is examined using the cockroach Periplaneta americana and the fruit fly Drosophila melanogaster as model species. The effects of Peram‐CAH‐I and ‐II on the activity of cockroach digestive enzymes in the gastric caeca and midgut are measured both in vivo and in vitro. The results show the activity of proteases, amylases and lipases in both parts of the gut: amylase activity is higher in the gastric caeca than in the midgut; lipase activity presents the opposite trend; and protease activity is similar in both organs. The applied hormones stimulate the activity of all digestive enzymes, although this stimulation is not uniform; AKHs affect enzymes selectively, and in some cases unequally, in the gastric caeca and midgut. No substantial differences between Peram‐CAH‐I and ‐II stimulation are recorded. The in vitro results demonstrate that AKH stimulates digestive enzyme activity directly. In agreement with the cockroach results, enzymatic activity in D. melanogaster larvae producing nonfunctional AKH is lower than that in the larvae with ectopically expressed Akh gene, where enzyme activity reaches or even exceeds that of the controls. Overall, the results demonstrate the active role of AKHs in the stimulation of digestive enzyme activity in insects.     

Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity

Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.     

Allocation strategies in crustacean stoichiometry: the potential role of phosphorus in the limitation of reproduction

1. Elemental composition (carbon : nitrogen : phosphorus, C : N : P) was analysed in eggs and juveniles of two crustaceans, Daphnia magna (Cladocera) and the crayfish Astacus astacus (Decapoda). Stoichiometry was also analysed for the carapace, muscle tissue, hepatopanchreas and gills of Astacus. 2. For both species the C : P ratio was significantly higher in eggs than juveniles, but there was a constant, homeostatic elemental ratio in eggs during embryogenesis (Astacus) and with different C : N : P in maternal food (Daphnia). 3. Differences in the stoichiometry of major tissue categories in Astacus suggest that there are distinct allocation strategies of elements to various somatic tissues as well as to reproduction versus somatic tissues overall. 4. There are strong ontogenetic shifts in the allocation of energy and elements in both species, as for crustaceans in general. During maturity there may be a trade‐off with regard to the allocation of C, N or P to somatic or reproductive tissue, and poor food quality (high C : P in food) could pose other constraints on reproductive capacity than does food shortage (low C). 5. Egg production may be at least as sensitive to low P as is somatic growth and could result in a marked decrease in overall population growth rate more severe than would be expected from individual growth rate alone.     

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, <Emphasis Type="Italic">Cherax destructor</Emphasis> and <Emphasis Type="Italic">Engaeus sericatus</Emphasis> (Astacidae,Parastacidae)

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81–82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-β-1,4-glucanase, β-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-β-1,4-glucanase and β-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-β-d-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.     

A field study on the physiology of digestion in the Antarctic krill, Euphausia superba, with special regard to chitinolytic enzymes

Endo- and exochitinase activities were determined in the stomachand midgut gland of the Antarctic krill, Euphausia superba.along a transect west of the Antarctic Peninsula. Activitieswere compared with the digestive enzymes protease, cellulase(1,4-ß-D-glucanase) and laminarinase (1,3-ß-D-glucanase)The chlorophyll and protein contents in the surface water ofthe corresponding stations were determined. Enzyme activitieswere characterized by high individual and spatial variations.Chitinolytic activity in the stomach correlated well with alldigestive enzymes investigated. In the midgut gland, a correlationwith cellulase and laminannase was evident. The amount of chlorophylla and phytoplankton protein in the surface water was not correlatedwith enzyme activity. Specific enzyme activity was higher inthe stomach than in the midgut gland. showing individual ratiosfor each enzyme. Elevated endochitinase activity in the stomachsuggests that chitinous food is digested to oligomers in thestomach, while the subsequent degradation to amino sugars occurspredominantly in the midgut gland.     

Cathepsin D-Like Aspartic Proteinase Occurring in a Maize Weevil,Sitophilus zeamais,as a Candidate Digestive Enzyme

The maize weevil, Sitophilus zeamais, is an insect pest infesting rice and corn seeds. We identified an aspartic proteinase (AP) digesting rice glutelin in the alimentary tract of S. zeamais. The mRNA encoding the AP (SAP1) was expressed in the larvae foregut and in the adult midgut. These results indicate that SAP1 is probably digestive enzyme of S. zeamais.     

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae)

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.     

Characterization and comparison of digestive proteinases of the Cortez swimming crab, Callinectes bellicosus, and the arched swimming crab, Callinectes arcuatus

Abstract. Protease activity in the midgut gland, gastric chamber, and gastric juice from the crabs Callinectes bellicosus and Callinectes arcuatus was characterized by several methods, confirming that the composition of digestive proteases is the same in the gastric juice and the midgut gland. Gastric juice was suitable for the identification and characterization of the proteinases trypsin and chymotrypsin. Such enzymes were presented as isotrypsins and isochymotrypsins. Proteinase composition evaluated by SDS-PAGE and substrate-SDS-PAGE showed differences between species, but not between gender. Proteinases were thermostable at 40°–50°C for 1 h and showed maximum activity at pH 6–8, making the use of digestive proteinases for evaluations of protein digestibility by the pHstat method possible. We propose using gastric juice as a source of digestive enzymes for in vitro studies of enzymes in digestibility assays and characterization procedures.     

Cellular localisation of aminopeptidase in the midgut of Rhodnius prolixus Stål (Hemiptera:Reduviidae) during blood digestion

Summary By use of the artificial substrate leucyl--naphthylamide, aminopeptidase was localised in the midgut cells of the haematophagous insect Rhodnius prolixus before and at various times up to 25 days after a meal of rabbit blood. The enzyme was primarily associated with the membranes of the microvilli, with extracellular membrane layers and with the lysosomes of the midgut cells. Aminopeptidase activity was also detected on the rough endoplasmic reticulum and at the periphery of intracellular storage vesicles. The absence of aminopeptidase on the microvilli of the crop supports the conclusion that the crop is not involved in the digestion of blood-meal proteins and that protein digestion is restricted to the intestine. The sites of localisation are in accordance with models for the spatial separation of digestive enzymes in the midgut of several non-haematophagous insects, and this suggests that aminopeptidase plays a major role in the terminal digestion of the blood meal. The changes in enzyme localisation during the digestive period correlate with previously described cycles of digestive-enzyme activity and changes in midgut ultrastructure. A model for blood protein digestion in R. prolixus is described.     

An hypothesis of the mechanism controlling proteolytic digestive enzyme production levels in Stomoxys calcitrans

Flies fed a human blood meal and sacrificed 9 h later were assayed to give information on unfed fly weight, meal weight, total midgut protein, total midgut proteolytic activity, anterior midgut protein, anterior midgut proteolytic activity, posterior midgut protein, and posterior midgut proteolytic activity; correlation coefficients were calculated for all pairings of these parameters. Posterior midgut protein showed a positive correlation with posterior midgut proteolytic activity and on this evidence it is concluded that proteolytic digestive enzyme secretion in the midgut of Stomoxys calcitrans is controlled by a secretogogue mechanism.It is proposed that the only direct stimulus the food supplies in the control of digestive enzyme production is that for digestive enzyme release from the production cells. It is also proposed that the basis of the secretogogue mechanism is that digestive enzymes are produced in direct proportion to the quantities of amino-acids available for their synthesis and that this is a consequence of the quantities of amino acids released from the food during digestion.     

Vorkommen und Eigenschaften der Carbonsäureesterasen im Hepatopankreas und Magensaft des Flußkrebses Astacus astacus (L.) und Cambarus affinis (Say)

Zusammenfassung Es wurden die Esteraseaktivitäten in wäßrigen Mitteldarmdrüsenextrakten und in Magensaft(MS)-Sammelproben mehrerer Flußkrebse [Astacus astacus (L.) und Cambarus affinis (Say)] quantitativ bestimmt. Die höchsten Aktivitäten enthält das Astacus-Hepatopankreas (HP), das, gemessen an der Hydrolyse der optimalen Substrate, 10- bis 23mal aktiver ist als das Cambarus-HP und 171-bis 247mal aktiver als der Magensaft beider Krebsarten. Das pH-Optimum der HP-Esterase-Wirkung liegt zwischen pH 8 und 9, das der sehr gering aktiven Magensaftesterase bei pH 6,0 (für Tributyrin) bzw. 7,5–8,5 (für Phenylacetat und-butyrat). Die unter Verwendung von 21 Estersubstraten näher untersuchte Substratspezifität der Astacus-HP-Esterase ergab eine Affinität für aliphatische und Phenyl sowie Naphthyl-Ester der C₃- bis C₅-Fettsäuren. Die Esterase ist relativ thermostabil, durch E 600, NaF und Eserin stark hemmbar und zeigt keine Cholataktivierung. Durch Sephadex-Gelfiltration wird die HP-Esterase in zwei Aktivitätsgipfel aufgetrennt (K _d -Werte von Sephadex G-200: 0,083 bzw. 0,5–0,52), die die gleiche Substrat und Inhibitor-Spezifität aufweisen. Im Agargelelektropherogramm des HP sind vier Esterasezonen, im Pherogramm des MS nur eine schwache Esterasezone mit -Naphthylbutyrat als Substrat nachweisbar. Die MS-Esterase besitzt die gleiche Wanderungsgeschwindigkeit wie die am weitesten anodisch wandernde schwächste HP-Esterasebande.

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Occurrence and properties of the carboxylic esterases in hepatopancreas and gastric juice of the crayfish Astacus astacus (L.) and Cambarus affinis (Say)

Summary The esterase activities of hepatopancreas extracts and gastric juice from the crayfish Astacus astacus (L.) and Cambarus affinis (Say) were determined quantitatively. Astacus-hepatopancreas contains the highest activities. It is 10 to 23 times more active than Cambarus-hepatopancreas and 171 to 247 times more active than gastric juice of the two species. The pH-optimum of the hepatopancreas esterase action was found to be between pH 8 and 9, that of the very weak gastric juice esterase at pH 6.0 and pH 7.5 to 8.5 with tributyrin and phenyl acetate and butyrate respectively. Substrate specifity studies with 21 ester substrates showed that the Astacus-hepatopancreas esterase posseses a high affinity towards phenyl, naphthyl and aliphatic esters of C₃- to C₅-chain fatty acids. The esterase is relative thermostabil, E 600, NaF, and eserine sensitive and was not activated by cholate. By means of Sephadex gelfiltration the hepatopancreas esterase was separated in 2 fractions (K _d -values on Sephadex G-200: 0,083 and 0.5 to 0.52 respectively) with identical substrate and inhibitor specificity. About four zones of esterase activity may be detected in hepatopancreas extracts and only one weak esterase zone in gastric juice by agar-gel electrophoresis (substrate: -naphthylbutyrate). The electrophoretic mobility of the gastric juice esterase is identical with that of the fastmoving anodal and weakest esterase component of hepatopancreas.

     

Digestive α-amylase activity in Aelia acuminata L. (Hemiptera: Pentatomidae)

Activity of α-amylase was revealed in the midgut and salivary glands of the wheat and barley pentatomid pest, A. acuminata. The activity was determined in salivary gland more than those in midgut. Optimal activity of the enzyme occurred at 40°C. Optimal pH activity in salivary gland (pH = 6) was more than those in the midgut (pH = 4.5). pH stability analysis of the enzyme showed that the enzyme is more stable at slightly acidic pHs than those at acidic and alkaline pHs. However, α-amylase is more stable at acidic pH in long period of time. Temperature stability analysis determined the enzyme was remarkably active over a broad range of temperature (5–40°C). α-Amylase activity was decreased after addition of MgCl₂, Tris, Triton X-100, CuSO₄, SDS, urea and CaCl₂. The salts NaCl and KCl increased the enzyme activity from midgut and salivary glands. Zymogram analysis of midgut and salivary gland extract showed at least two bands of amylase activity in the midgut and salivary glands.     

A laboratory study on digestive processes in the Antarctic krill,Euphausia superba,with special regard to chitinolytic enzymes

Feeding experiments of 9, 14 and 20 days duration were carried out on the Antarctic krill, Euphausia superba. Two groups were fed with the chitinous diatom Cyclotella cryptica and the non-chitinous green algae Dunaliella bioculata, respectively. A control group remained unfed. The time courses of the activities of endo- and exochitinase in the stomach and the midgut gland were compared with those of the digestive enzymes protease, cellulase (1,4-β-d-glucanase) and laminarinase (1,3-β-d-glucanase). Specific activities of all enzymes were higher in the stomach than in the midgut gland. Characteristic time courses of activity were evident after 4 days. In starved animals, enzyme activities decreased to a minimum after 4 days and recovered within 14 days to initial values. In the stomach, the activities of endo- and exochitinase increased when krill were fed on Cyclotella. For animals fed with Dunaliella, activities stayed constant or decreased slightly. The results confirm chitinases as digestive enzymes and, therefore, the capability of krill to utilize various food sources. Accepted: 4 October 1998     

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood-feeding termite, Reticulitermes speratus (Kolbe): possible digestion of symbiont bacteria transferred by trophallaxis

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood‐feeding termite, Reticulitermes speratus (Kolbe) (Isoptera, Rhinotermitidae) were studied to examine the possibility that termites digest symbiont bacteria transferred by trophallaxis. Total lysozyme activity was found predominantly in the salivary gland and to a minor extent in the digestive tracts. However, specific lysozyme activity was high in the foregut as well as in the salivary gland. The similarity of the lysozyme pH profile of the salivary gland and of the foregut suggested that the foregut lysozyme came from the salivary gland. Major protease activity having the optimum pH of 7.5 was found in the midgut. Total free amino acid amount and concentration in the midgut was higher than elsewhere in the digestive tract. The possibility that lysozyme secreted from the salivary gland into the foregut digests hindgut bacteria transferred by trophallaxis was discussed.