Antioxidant enzymes in the digestive gland of the common musselMytilus edulis

Antioxidant enzymes function to remove deleterious reactive oxygen species, including the superoxide anion radical and H₂O₂. Subcellular distributions and optimal and other properties of catalase (EC. 1.11.1.6), superoxide dismutase (SOD; EC. 1.15.1.1), selenium-dependent glutathione peroxidase (Se-GPX; EC. 1.11.1.9) and total glutathione peroxidase (GPX) activities were determined in the digestive gland of the common musselMytilus edulis L. by spectrophotometric and cytochemical/electron microscopic (catalase) techniques. Assay conditions for Se-GPX and total GPX activities were determined which optimized the difference between the non-enzymic and enzymic rates of reaction. General peroxidase activity (guaiacol as substrate) (EC. 1.11.1.7) was not detectable in any subcellular fraction. Catalase was largely, if not totally, peroxisomal, whereas SOD and GPX activities were mainly cytosolic. Distinct mitochondrial (Mn-SOD) and cytosolic (CuZn-SOD) SOD forms were indicated. Catalase properties were consistent with a catalase, rather than a catalase-peroxidase. The pH-dependence and temperature-dependence of GPX activity were different with H₂O₂ or CHP as substrate, and these and other observations indicate the existence of a distinct Se-GPX. Under saturating or optimal (GPX) assay conditions, the apparent Michaelis constantsK _m (mM) were: catalase, 48 to 68 (substrate, H₂O₂); Se-GPX, 0.11 (H₂O₂) and 2.0 (glutathione); and total GPX, 2.2 (eumene hydroperoxide) and 1.2 (glutathione). Calculated catalase activity was 2 to 4 orders of magnitude greater than Se-GPX activity over an [H₂O₂] of 1 to 1000 M. The results are discussed in relation to theoretical calculations of in vivo oxyradical production and phylogenetic differences in antioxidant enzyme activities.     

Chitinolytic enzymes in the digestive system of marine fishes

Chitinase, exo-N-acetyl--D-glycosaminidase (NAGase) and lysozyme activities were assayed in the digestive tract of 6 species of marine fishes: Myxine glutinosa (cyclostome), Chimaera monstrosa (holocephalan), Squalus acanthias, Etmopterus spinax, Raja radiata (elasmobranchs) and Coryphaenoides rupestris (teleost). Strong chitinase activity was found in the gastric mucosa of the elasmobranchs (S. acanthias, E. spinax and R. radiata) and the teleost (Coryphaenoides rupestris). A remarkably high chitinase activity occurred in the pancreas of the stomachless holocephalan fish Chimaera monstrosa. NAGase activity was strong in the digestive tract of all species. It could be concluded that marine fishes with diets consisting largely of chitinous invertebrates may display high chitinase and NAGase activities in their digestive system; however, only low chitinase activity was found in the intestine of the cyclostome Myxine glutinosa. Coryphaenoides rupestris gastric mucosa chitinase had one optimum activity at pH 1.25, whereas S. acanthias chitinase had two optima, at pH 1.6 and 3.6. The NAGase pH-activity curves from S. acanthias and R. radiata gastric mucosa displayed similar optima, at pH 4.5 and 4.25 respectively. Chimaera monstrosa pancreatic chitinase had a very strong optimum around pH 8 to 10, and one less strong at pH 3. These enzyme activities could not be separated by gel filtration or isoelectric focusing. The pI (isoelectric point) was approximately 4.9 for both enzymes. The molecular weight of the C. monstrosa pancreatic chitinase was estimated to be approximately 43 000. Lysozyme activity was absent or extremely weak in the material studied.     

The production and secreton of digestive enzymes in the purple seastar Pisaster ochraceus

The purple seastar Pisaster ochraceus contains clearly measurable protease and amylase activity. Centrifuged supernatants of pyloric caeca homogenates undergo a spontaneous threefold increase in protease activity when incubated under toluene for 50 h at 25°C. Amylase activity remains nearly constant over this period. Bovine trypsin at a 1 to 100 ratio (trypsin to supernatant protein) induces a twofold increase in protease activity over that of the control supernatant while having virtually no effect on amylase activity over the control. The data indicate a specific interaction of trypsin with a protease zymogen rather than a conspecific hydrolysis of membrane components or vesicles by trypsin. Two percent Triton X-100 used as a diluent in place of distilled, deionized water causes a sevenfold increase in protease activity and a twofold increase in amylase activity in pyloric caeca supernatants. The use of Triton as a diluent in the preparation of a stomach-tissue supernatant allows quantitative measurement of both protease and amylase activity in that tissue.     

A study on the activities of a few free radicals scavenging enzymes present in five roadside plants

The road side plants are continuously exposed to the high levels of oxides of nitrogen and sulphur dioxide, emitted from automobile. Resistance to automobile exhaust pollution was studied with Nerium indicum Mill, Boerhaavia diffusa L., Amaranthus spinosus L., Cephalandra indica Naud., and Tabernaemontana divaricata L., growing on the edges of Delhi Road, National Highway 2 (NH 2) near Dankuni, West Bengal. By analysing the activities of a few enzymes like superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and phenolic peroxidase, it appears that among the five plants examined,Amaranthus and Cephalandra are equipped with a very good scavenging system to combat effects of air pollution.     

Ultrastructure of the digestive gland of larval and adult stages of the sacoglossan Elysia patagonica

Chloroplast retention in the sacoglossan gastropod Elysia patagonica Muniain and Ortea, 1997 was investigated. Intact chloroplasts of the green algae Bryopsis plumosa (Chlorophyta: Bryopsidacea) were observed by transmission electron microscopy in vacuoles of the digestive cells of benthic adults starved for 20 days. Intact chloroplasts from the microalgal Nannochloropsis sp. (Heterokontophyta: Eustigmatophycea) were found in the digestive cells of veliger larvae feeding for 8 days. Similar digestive cells were observed in adults and planktonic larvae of E. patagonica. The retention ability is confirmed in both stages.     

Activity of the principal digestive enzymes in the detritivorous apodous holothuroid Leptosynapta galliennei and two other shallow-water holothuroids

Digestive ability by means of hydrolytic enzyme activities was studied in Leptosynapta galliennei (Herapath, 1865) and two other shallow-water deposit-feeding holothuroids: L. inhaerens (O. F. Müller, 1776) and Holothuria (Panningothuria) forskali (Delle Chiaje, 1828). The holothuroids were collected throughout the year at Roscoff (Britanny, France) from 1980 to 1985, except for L. inhaerens, which was collected in Galway Bay (Ireland) in June/July 1980. Using 25 substrates, the activity of 21 enzymes were recorded to detect hydrolysis of esters (5 substrates), glycosidic bonds (12 substrates) and peptide bonds (8 substrates). The activities of the homogenates of various parts of the gut as well as the digestive juice were measured. Esters and relatively long-chained fatty acids (up to C₁₄) were hydrolysed. Significant hydrolysis of naturally occurring disaccharides and starch (reserve carbohydrate) occurred. The holothuroids were also able to digest hydrolysis products of cellulose and chitin. This may also indicate a capacity to digest the products of complex compounds such as glycolipids or glyco-proteins. Exopeptidases capable of hydrolyzing peptides were present, but endopeptidases able to digest proteolytic chains were not. Most enzymatic activities occurred in all parts of the gut, but some were strictly localized, usually to the anterior intestine. Extracellular digestion of fatty acids, starch and saccharose occurred only in this latter digestive segment. Disaccharidases and peptidases are linked to the plasma membrane of enterocytes. Enzymatic activities associated with lysosomes occur throughout the gut. The diet of shallow-water holothuroids is omnivorous, with a marked preference for food of vegetable origin. Annelids and holothuroids possess a similar capacity to hydrolyse most types of bonds, including peptide bonds. The chemical nature of detritus and its relationship to the nutritional requirements of detritivores must be defined, and gut-retention time ascertained, to discover if different digestive strategies are developed by different detritivores to exploit the same food source in a given environment.     

Lipids in the digestive gland and the gonad of immature and mature Sepia officinalis (Mollusca: Cephalopoda)

The lipid content of the digestive gland and the gonad of cuttlefishes (Sepia officinalis L.) caught off the French coasts in 1979–1980 was determined and analysed by thinlayer chromatography. The total sterol and phospholipid levels were also determined. The results from immature and mature females and males were compared in an attempt to correlate variations of the lipids with sex or state of gonad maturation. The fluctuations in the lipid level of the digestive gland, which acts as a storage organ, seemed far better related to the diet than to sex or sexual maturity. In the gonads, the lipids were few and seemed to be mostly membrane constituents. However, the increase of phospholipids in the mature ovary could be related to yolk synthesis.     

Effects of lead exposure on redox status,DNA and histological structures in Venus verrucosa gills and digestive gland

ABSTRACT

Lead (Pb) is one of the most toxic heavy metals that affect the physiological status of aquatic organisms. The present investigation evaluated the possible toxic effect of lead chloride (PbCl₂) on biomarkers responses, DNA damage and histological alterations in Venus verrucosa gills and digestive gland. Three concentrations of PbCl₂ (D1:1µgL^?1, D2: 10µgL^?1 and D3: 100µgL^?1) were chosen for V. verrucosa exposure during six days. At the end of the trial, it was found that Pb tended to accumulate in both gills and digestive gland in a dose-dependent manner. However, gill tissues exhibited the highest metal burden. Our results showed an increase of malondialdehyde, protein carbonyls and advanced oxidation protein product levels in both organs following PbCl₂exposure. The induction of both non-enzymatic and enzymatic antioxidant systems; as well as the decrease of the acetylcholinesterase activity and degradation of DNA structure was recorded in the gills and digestive gland. The histopathological alterations observed in gills (disruption of lamellas and cilia filaments?…) and digestive gland (lumens occlusion, necrosis and fibrosis) confirmed the aforementioned results. Our data highlighted the short-term toxicity effects of PbCl₂ on V. verrucosa and pointed out a high sensitivity of gills towards this metal.     

Molecular association of Cu,Zn, Cd and 210Po in the digestive gland of the squid Nototodarus gouldi

In July 1985, very fine burrows extending at least to a sediment depth of 50 cm were found in sediments of the Vöring-Plateau, Norwegian continental slope, at depths of 1 261 to 1 969 m. Such burrows have been described from other areas of the oceans, but until now the producers have been unknown. In the box-cores from Vöring-Plateau sipunculans of the genus Golfingia, subgenus Nephasoma, were identified as inhabitants and producers of these Lebensspuren, which correspond with the fossil ichnogenus Trichichnus.     

Evidence that lipid can be digested by the dumpling squid Euprymna tasmanica, but is not stored in the digestive gland

The aims of this study were to assess quantitatively the enzymatic ability of squid to digest lipids and the ability of the digestive gland to accumulate lipid classes associated with storage. This was achieved through two manipulative experiments using the dumpling squid, Euprymna tasmanica. Firstly, we measured lipase activity and determined the presence and location of lipid vacuoles within the digestive gland; secondly we identified and quantified lipid classes in the digestive gland. Given the levels of lipase activity, we provided evidence for the first time that a squid species is capable of digesting lipid at levels comparable to invertebrates known to use dietary lipid. A poor relationship between feeding activity and lipase secretion suggests that enzyme production is continuous. The second experiment found no evidence that lipid was stored in the digestive gland; most of the lipid present in the gland was either structural or a dietary by-product. The implication of these findings is that for this species lipid is most probably being immediately digested and used for growth and reproduction rather than being stored in the digestive gland. We consider that the role and storage of lipid is likely to vary among different cephalopod species, but not predictably as function of their lifestyle. Therefore, potential locations for lipid storage, other than the digestive gland, need to be considered and using changes in the relative size of the digestive gland as a measure of condition needs to be interpreted with care.     

Effects of chronic copper exposure on the green mussel Perna viridis

The effects of chronic copper exposure on growth and physiological responses of the green mussel Perna viridis were investigated by exposing the mussels to 50 μg l⁻¹ Cu for 3 mo at 17 and 25 °C. These temperatures represent, respectively, the winter and summer seawater temperatures in Hong Kong. Differences in the level of response between mussels exposed for 3 mo to 50 μg Cu l⁻¹ generally increased with duration of exposure. The tissue concentration of copper had increased by 280 and 450% after 3 mo exposure at 17 and 25 °C, and growth performances were reduced, with the 25 °C sets suffering from larger negative impact of copper in most responses. The inhibitory effects of copper on production of the various body components generally followed the order linear shell growth (greatest) > tissue production > byssus production > shell production. There were also decreases in the condition index (43 and 35% reductions at 17 and 25 °C), clearance rates (10.3 and 18.5%), faeces production (11 and 16.3%), assimilation efficiency (6.8 and 9.2%) and oxygen consumption rate (12.8 and 24.8%). In contrast, the organic content of the faeces (9.2 and 13.2% increases at 17 and 25 °C) and rate of ammonia excretion (21 and 28.6%), increased upon chronic copper exposure. Many of the responses (e.g. changes in tissue copper content, body dry wt, shell organic content, clearance rate and oxygen consumption rate) exhibited fluctuating levels of impact during prolonged copper exposure, while others (e.g. faecal production rate, assimilation efficiency, tissue organic content) demonstrated steady decreasing trends with increasing exposure time. Received: 17 September 1999     

Uptake and depuration of cesium in the green mussel Perna viridis

 The accumulation and depuration of Cs in the green mussels (Perna viridis) commonly found in the subtropical and tropical waters were studied under the laboratory conditions using radiotracer techniques. Following an initial rapid sorption onto the mussel's tissues, uptake of Cs exhibited linear patterns over a short exposure time (8 h) at different ambient Cs concentrations. The concentration factor was independent of ambient Cs concentration. The calculated uptake rate and initial sorption constant of Cs were directly proportional to the ambient Cs concentration. The calculated uptake rate constant from the dissolved phase in the mussels was as low as 0.026 l g⁻¹ d⁻¹. Uptake rates of Cs in the mussels were inversely related to the ambient salinity. Uptake increased about twofold when the salinity was reduced from 33 to 15 ppt. The effect of salinity on Cs uptake was primarily due to the change in ambient K⁺ concentration. The uptake rate decreased in a power function with increasing tissue dry weight of the mussels, although the initial sorption was not related to the mussel's body size. The efflux rate constant of Cs in the mussels was 0.15 to 0.18 d⁻¹, and was the highest recorded to date among different metals in marine bivalves. The efflux rate constant also decreased in a power function with increasing tissue dry weight of mussels. A simple kinetic model predicted that the bioconcentration factor of Cs in the green mussels was 145, which was higher than measurements taken in their temperate counterparts. The bioconcentration factor also decreased in a power function with increasing tissue dry weight of mussels. Received: 27 October 1999 / Accepted: 16 June 2000     

Changes in the digestive gland of Euphausia superba during short-term starvation: lipid class,fatty acid and sterol content and composition

During a period of short-term (19 d) starvation, total lipid in the digestive gland of Euphausia superba Dana decreased from 21 to 9% dry weight. Total lipid per digestive gland decreased significantly during starvation compared to Day 0 individuals, falling from 1960 (±172) to 385 (±81) g. Polar lipid was the major lipid class utilised during starvation, falling from 1510 (±225) to 177 (±46) g per digestive gland (76 to 45%). Absolute levels of triacylglycerol fell from 300 (±41) to 76 (±5) g; however, relative levels remained unchanged. The relative level of free fatty acid increased significantly with starvation (4 to 39%) with absolute levels ranging from 79 (±1) to 156 (±20) g per digestive gland. Absolute levels of all fatty acids per digestive gland declined continually until the end of the starvation period. The long-chain polyunsaturated acids eicosapentaenoic (20:53) and docosahexaenoic (22:63), decreased with starvation from 37 to 26% and 15 to 10%, respectively whereas the saturated fatty acid, palmitic acid (16:0), increased from 15 to 20%. Cholesterol, the major sterol in this organ, increased from 17 (±20) to 44 (±13) g per digestive gland by Day 3, and by Day 19 had returned to levels found in the digestive gland of Day 0 individuals. Desmosterol followed a similar pattern to cholesterol, increasing from 3 (±1) g per digestive gland on Day 0 to 11 (±4) g on Day 3, and falling to 2 (±1) g on Day 19. Other sterols in the digestive gland, predominantly of algal origin, fell from the levels found in Day 0 individuals to near zero amounts by Day 6. The digestive gland of E. superba plays a dynamic role during shortterm starvation in terms of lipid content and composition. The relative levels of polar lipids, free fatty acids and cholesterol in the digestive gland may provide reliable indices of the nutritional condition of E. superba in the field. Sterols in the digestive gland are indicative of recent dietary composition of krill, and may also be used to quantify dietary input from individual phytoplanktonic species.     

Predator shoaling moderates the confusion effect in blue-green chromis,Chromis viridis

Summary In experiments, blue-green chromis [Chromis viridis (Cuvier 1830)] were fed on either scattered or aggregated swarms of brine shrimp (Artemia sp.). Ten runs with each prey dispersion treatment were performed with shoals of one, two, five and ten chromis. The mean lag in reaching peak feeding rate for fish fed on aggregated prey was significantly shorter in the larger chromis shoals. In contrast, with the scattered treatment all such lags were similar and very short. As foraging proceeded, higher feeding rates were observed in the larger feeding shoals, regardless of prey dispersion. Prey capture success (i.e. the rate of retention of intercepted prey) declined with time, but was significantly higher in groups of ten fish. Two main conclusions emerge. Firstly, grouping facilitated initiation of feeding by individuals preying on concentrated swarms and reduced the delay in reaching a maximum feeding level. This may have been due to a suppression of the confusion effect through reduced reliance upon vigilance. Secondly, reduced vigilance allowed larger shoals of chromis to feed effectively over more extended periods. Trends of increasing shoal cohesion and decreasing prey retention rate with time were consistent with a postulated increase in antipredator vigilance with declining feeding motivation.     

Induction of anti-predator responses in the green-lipped mussel Perna viridis under hypoxia

Hypoxia due to the over enrichment of waters by nutrients is becoming a global problem. In mussels, enhanced byssus thread production is an important adaptation to the presence of crustacean predators and to energetic hydrodynamic regimes. Thread production is an energy-consuming process, so this study used the green mussel Perna viridis (L.) to examine the response to predator exposure combined with hypoxia. Hypoxia is common in sheltered bays in Hong Kong, and the mussels were collected in one such bay, Lok Wo Sha (latitude/longitude: 22o18′ N/114o10′ E) in January, 2009. The predator used in the experiments was the swimming crab Thalamita danae. Oxygen concentrations used in the 48-h experiments ranged from hypoxic to normoxic (1.5 ± 0.3 mg l⁻¹, 3.0 ± 0.3 mg l⁻¹ and 6.0 ± 0.3 mg l⁻¹). Fewer byssus threads which were also shorter and thinner were produced at reduced oxygen levels, no matter if the predator was present or not; the frequency the mussels shed stalks was also lower. Mussels exposed to the predator, however, have enhanced byssus thread production at all oxygen levels when compared with the control. This has highlighted the significance of anti-predator responses for the survival of individuals even under a stressful environment in which energy supply is limited by aerobic metabolism. Interactive effects between oxygen level and predator exposure were observed for the byssus thread production (frequency of shed stalks, mean byssus thread length, cumulative byssus thread volume), with values obtained at 1.5 and 3.0 mg O₂ l⁻¹ being statistically indistinguishable for the control group without predator but not for the predator group. The lack of differences in the byssus thread production at lower oxygen levels in the absence of predator may indicate the minimum amount of byssus that is required for settlement on a substrate.     

Larval settlement in the echiuran worm Bonellia viridis: Settlement on both the adult proboscis and body trunk

It is widely documented that in Bonellia viridis Rolando (Echiura), the larvae settle on the proboscis of the adult female and differentiate into males. This study shows that although settlement on the proboscis is preferred, the larvae can also settle on the body trunk of the adult and differentiate into males. Aqueous extracts of homogenized trunk tissues are as effective as those of proboscis tissues in masculinizing the larvae. The natural secretion of the female body trunk, which is produced when the worm is disturbed, has potent masculinizing properties. The preferential settlement on the probiscis may not result from a different chemical nature of the substratum.     

Studies on the carbohydrases in the digestive tract of the milkfish chanos chanos

Crude extracts from various regions of the digestive tract of pond grown milkfish were tested forttheir ability to catalyze the hydrolysis of various carbohydrates. The most active carbohydrases were those involved in the hydrolysis of -glucosidic bonds. Maltose, trehalose, dextrin, starch and glycogen were rapidly hydrolyzed in the presence of crude extracts from the intestines and the pyloric caeca. High amylase activity was observed in extracts from the intestines, pancreas, pyloric caeca and liver. The intestinal amylase had optimum activity at pH 6.2 and at a temperature of about 50°C. It was active at a chloride concentration of 10 to 40 ppt. The amylase activity in the intestines consistently peaked daily at about noon when the milkfish gut was full. In contrast, enzyme activity was significantly lower at 0030 hrs when the gut was empty. These results are consistent with earlier observations that the milkfish is a daytime feeder and suggest further that intestinal amylase secretion is in phase with the feeding activity of the milkfish. Although the fishes used in this study fed mostly on the naturally occurring algae in the ponds, no cellulase activity was detected in any region of the digestive tract. Less active carbohydrases that were detected include a -glucosidase and -galactosidase, both of which were of limited substrate specificity.SEAFDEC Contribution No. 71     

Studies on the digestive proteases of the milkfish Chanos chanos

The protease activity of crude extracts from various organs of the digestive tract of two groups of milkfish was determined. One group (Sample A) derived their food from ponds that had predominantly unicellular algae while the other group (Sample B) were reared on ponds dominated by the filamentous green algae Chaetomorpha brachygona. In general, crude extracts from Sample A fish had a higher protease activity than Sample B fish. In both samples, high protease activity was observed in crude extracts from the pyloric caeca, intestines and pancreas. Chymotryptic activity was observed in crude extracts of pancreas, intestines and pyloric caeca of both fish samples. Tryptic activity was, however, observed only in fish grown on unicellular algae. Experimental evidence suggests that a powerful trypsin inhibitor in Chaetomorpha brachygona may account for the absence of tryptic activity in all crude extracts of Sample B fish. The presence of this inhibitor may also explain the widely observed poor growth rate of milkfish reared on this natural food.SEAFDEC contribution no. 112     

Primary cell-culture of the digestive gland of the marine mussel Mytilus edulis: a time-course study of antioxidant- and biotransformation-enzyme activity and ultrastructural changes

The extent to which the American lobster, Homarus americanus (H. Milne-Edwards), utilizes estuarine habitats is poorly understood. From 1989 to 1991 we examined lobster movements in and around the Great Bay estuary, New Hampshire using tag/recapture and ultrasonic telemetry. A total of 1212 lobsters were tagged and recaptured at sites ranging from the middle of Great Bay, 23.0 km from the coast, to Isles of Shoals, 11.2 km offshore. Twenty-six lobsters equipped with ultrasonic transmitters were tracked for periods ranging from 2 weeks to >1 year. Most lobsters moved <5 km toward the coast, with those furthest inland moving the greatest distance. Lobsters with transmitters moved in a sporadic fashion, with residency in one area for 2 to 4 weeks alternating with rapid movement to a new location (mean velocity = 0.3 km d⁻¹, 1.8 km d⁻¹ max.). Site of release influenced distance moved, but there was no significant relationship between lobster size and distance traveled, days at large, or rate of movement. Most movement into the estuary occurred in the spring, while during the remainder of the year there was a strong tendency to move downriver, toward the coast. These seasonal migrations of estuarine lobsters may enhance their growth and survival by enabling them to avoid low salinity events in the spring and fall, and to accelerate their growth in warmer estuarine waters during the summer. Received: 26 January 1996 / Accepted: 22 January 1999