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.     

Aspartic proteinases in the digestive tract of marine decapod crustaceans

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.     

Digestive proteases in the midgut gland of the atlantic blue crab,Callinectes sapidus

• 1.1. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.
• 2.2. Trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase activities were found and quantified.
• 3.3. Activity against Succinyl-(Ala)₃-nitroanalide was also found. This as yet unidentified enzyme has a mol. wt of about 26,000 and has elastolytic activity.

     

Digestive enzymes present in crustacean feces as a tool for biochemical, physiological, and ecological studies

The effect of food composition on the digestive system of Penaeus vannamei shrimp was used to determine the suitability of feces for analysis of class, type, composition of digestive proteinases, and whether alterations in the digestive gland are mirrored in feces composition. Enzymes recovered from feces and the midgut gland of white shrimp P. vannamei were used for comparison purposes. Three groups of shrimp were assembled: two groups fed two different brands of commercial feeds (PI and SC) with different content of protein, and the last group fed 50% PI feed and 50% thawed giant squid. Composition of proteinases in the midgut gland and feces were identical, and trypsin and chymotrypsin paralogues were identified in both samples by substrate-electrophoresis. Total proteolytic, trypsin, and chymotrypsin enzyme activities were higher in both samples from organisms fed SC, than in the other two groups. In the hepatopancreas, trypsin activity was ∼30% higher in SC fed group. Final average weights of shrimp were close in three groups, but hepatopancreas weight was 20% higher in the SC group. The degree of protein hydrolysis (DH) in vitro for the SC and PI was evaluated by the pH-stat method, using enzymes from feces and hepatopancreas of each group. The DH of food was no different, but it was affected by enzyme source, hepatopancreas extract (HPE) or feces extract (FE). DH was always higher when FE was the enzyme source than when HPE was the source. The proposed methods for recovery of enzymes from shrimp feces can be applied to other crustaceans. Measurements were sufficiently sensitive to allow quantifying the effects of feed on digestion physiology and other ecological and physiological applications, without the necessity of killing specimens.     

Characterization of Proteases in the Digestive System of Spiny Lobster (<Emphasis Type="Italic">Panulirus interruptus</Emphasis>)

Enzymes responsible for digestion of food protein were evaluated and characterized in red lobster (Panulirus interruptus). Several tissues, organs, and body fluids were analyzed. The same composition of proteases was found in gastric juice, midgut gland, and intestinal contents. Using specific substrates and inhibitors, we indentified several isotrypsins and isochymotrypsins by gel electrophoresis. Protease activity was found at pH 3 and reduced by using pepstatin A. Operational variables of enzymes were characterized for management of future studies and potential biotechnologies. Types and activities of lobster digestive enzymes constitute background information to study the digestive abilities of the organism further, and will lead to understanding nutritional needs and feeding ecology, mainly because decapods display unique morphologic, metabolic, and behavioral changes during their life cycle. Also, such enzymes become alternative tools for use in biotechnologies.     

Restoration of the secretory activity of the digestive glands under high physical load

The time course of the restoration reactions of the secretory functions of the gastric glands were studied in patients differing in the level and specificity of daily physical activity. Dependence was found between the level and specificity of daily physical activity and reactivity of the secretory apparatus of the gastric glands during the recovery period after physical load. A high reactivity of the digestive gland activity is typical of subjects with a high daily physical load. Differences in the functional stability of the secretory mechanisms of various components of the gastric juice were found in different states: at rest, under physical load, and in the recovery period. The maximal stability of the secretory mechanisms of the digestive gland was marked in athletes trained to develop endurance (skiers). Heterochronism of the restoration reactions of the gastric secretion after physical loads was found. At the beginning, secretion of enzyme is restored, followed by acid composition of the digestive juices. An inverse relationship was found between the content of the blood levels of the digestive enzymes pepsinogen-1 and -2 and the concentration of proteolytic enzymes in gastric contents in subjects with various adaptation degrees to the physical load.     

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.     

Enzyme-forming function of digestive glands after administration of polypeptide C, copepsidyl and panintestine

Polypeptide C (molecular weight 2640 Dalton) extracted from artificial gastric juice), copepsidyl (containing high dosage of pepsin) and panintestine (polyenzyme drug) are studied for their effect on the activity of digestive enzymes of glandular gastric element pancreas and small intestine of rats. It is established that all mentioned drugs stimulate enzymogenesis in the analyzed organs. The activity of pepsin increases in homogenates of gastric mucosa, the activity of trypsin, total proteinases, carboxypeptidase and amylase grows in pancreas homogenates, and that of leucineaminopeptidase--in small intestine.     

The digestion of cellulose and other dietary components, and pH of the gut in the amphipod Gammarus pulex (L.)

SUMMARY. Gut extracts from Gammarus pulex hydrolysed native and other cellulose substrates in vitro. Digestive fluid cellulase is probably endogenous as cell-free fluid mediated cellulose hydrolysis, but no bacteria were isolated from the fluid which produced a detectable extra-cellular cellulase. There was no apparent digestion of plant cell walls during their passage along the digestive tract, which took about 5–7 h at 10°C. The pH sensitivities of the digestive enzymes and the pH of the various regions of the gut suggest that carbohydrate digestion occurs in the proventriculus, midgut glands and anterior midgut, but protein digestion may be largely limited to the posterior midgut. The pH of the digestive fluid was altered slightly, but significantly, by the consumption of different natural and artificial test diets and by starvation. The most probable reason for the non-digestion of plant cell-walls is the lack of necessary enzymes other than cellulase. The role of cellulase may be confined to digesting the many small, non-cellular particles which are present in the gut.     

Dana swimming crab growth Callinectes danae (Decapoda: Portunidae) from Margarita Island, Venezuela

Callinectes danae is a common species captured with crab traps in nearby areas of coastal lagoons in Margarita Island. Although its considerable economic importance as a fishery resource, few studies have been done on population dynamics and its fishery potential in local coastal environments to support decision making in fishery administration. We present growth pattern details of Callinectes danae to better estimate its population size and exploitation feasibility. For this, we analyzed a total of 3 623 specimens that were monthly captured in crab pots by artisanal fishermen in Las Marites lagoon, from October 2007 to September 2008. The length-weight ratio was determined, and growth parameters estimated from both length and weight curves of the von Bertalanffy model. The general sex ratio showed no significant difference between males and females (chi2 = 0.04, p > 0.05). However, values of slopes b between males and females were significantly different (t(s) = 2.75, p < 0.05), as well as intercepts a (t(s) = 2.44, p < 0.05). Thus, the length-weight ratio was determined separately: W = 7.48e(-5)*L(2.98) for males and W = 1.21e(-4)*L(2.87) for females, indicating a negative allometric growth in both sexes. Growth parameters were established as: L(infinity) =134.80mm, W(infinity) = 166.04g and k = 0.86/yr for males; L(infinity) = 122.35mm, W(infinity) = 118.45g and k = 0.63/yr for females. Lifespan was estimated at 3.05 years for males and 4.24 years for females. We concluded that Callinectes danae is a species with short lifespan and moderately rapid growth. The coefficient of variation values (CV), of the phi-prime growth performance index (?'), showed a different growth pattern compared to those obtained in other regions. We propose that a management strategy will be the periodical review of the minimum capture size for fishing area, after the great variability found in growth parameters.     

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.     

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.     

Anatomy and physiology of the swimming leg musculature in the blue crab,Callinectes sapidus

1) The anatomy and neurophysiology of muscles moving the fifth leg of Callinectes sapidus are described. Innervation of the muscles was studied using intracellular stimulating and recording techniques.

2) The movement of the leg is controlled by four sets of muscles composed of two, three, or four bundles each. The bundles which make up each muscle shared innervation with other muscle bundles within a functional muscle group. Muscle fibers characteristically displayed both “fast” and “slow” responses to stimulation.

3) Possible functional evolution of the neural changes which occurred with the morphological adaptations for swimming in C. sapidus is discussed.     

Salinity-sensitive alkaline phosphatase activity in gills of the blue crab,Callinectes sapidus Rathbun

A levamisole-sensitive (K_i = 0.72 mM) alkaline phosphatase (pH optimum 9.1) and a levamisole-insensitive alkaline phosphatase (pH optimum 7.1) are present in gills of the blue crab Callinectes sapidus. Both enzymes are distinct from ouabain-sensitive ATPase. Specific activity for either phosphatase is greatest in the acinar tissue, which lines the branchial vessels. Histochemical localization of the enzymes confirmed this distribution. Activity of levamisole-sensitive alkaline phosphatase is affected by acclimation salinity. V_max of the levamisole-sensitive alkaline phosphatase is greater in high-salinity crabs than in low-salinity crabs; apparent K_m is not significantly different. The levamisole-sensitive alkaline phosphatase associated with the acinar tissue lining the branchial vessels may modulate the osmoregulatory response in blue crabs.     

Molt-inhibiting hormone immunoreactive neurons in the eyestalk neuroendocrine system of the blue crab, Callinectes sapidus

The production of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated by a neuropeptide, molt-inhibiting hormone. It is generally agreed that molt-inhibiting hormone is produced and released by the eyestalk neuroendocrine system. In the present study, immunocytochemical methods were used to detect molt-inhibiting hormone immunoreactive neurons in eyestalk ganglia of the blue crab, Callinectes sapidus. The primary antiserum used was generated against molt-inhibiting hormone of the green shore crab, Carcinus maenas. A preliminary Western blot analysis indicated the antiserum binds molt-inhibiting hormone of Callinectes sapidus. Using confocal and conventional immunofluorescence microscopy, molt-inhibiting hormone immunoreactivity was visualized in whole mounts and thin sections of Callinectes sapidus eyestalk ganglia. Immunoreactivity was detected in 15-25 neurosecretory cell bodies in the medulla terminalis X-organ, their associated axons and collateral branches, and their axon terminals in the neurohemal sinus gland. The cellular organization of molt-inhibiting hormone immunoreactive neurons in blue crabs is generally similar to that reported for other crab species. The combined results suggest the cellular structure of the molt-inhibiting hormone neuroendocrine system is highly conserved among brachyurans.     

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48?h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.     

Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.     

Antipeptide antibodies for detecting crab (Callinectes sapidus) molt-inhibiting hormone

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab (Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.     

Restoration of secretory activity of digestive glands in conditions of acute hyperkinesis at persons with different levels of motor activity

The dynamics' features of restoration reactions of the secretory function of gastric glands have been studied at patients with differences in the level and specificity of daily physical activity. The dependence between the level and characteristics of daily physical activity and reactivity of the secretory apparatus of the gastric glands in the recovery period after the muscle load has been established. The high reactivity of the secretory activity of digestive glands is typical for individuals with high level of daily physical activity. The differences of the functional stability of the secretion's mechanisms of various components of gastric juice under the influence of physical exercise and in recovery have been revealed. The greatest stability of the secretory mechanisms of digestive glands has been discovered at athletes practicing with the development of endurance--at skiers. Heterochronous reducing reactions of gastric and pancreatic secretion after physical stress have been defined. The reduction of secretion's mechanisms of gastric juice ingredients and the electrolyte and acid composition of digestive juices isn't simultaneous: the first order is for ferment's secretion. The inverse relationship between the content of digestive enzymes pepsinogen-1 and -2 in blood's serum and the concentration of proteolytic enzymes in the gastric content has been found at persons with various degrees of adaptation to the muscular tension.