Seasonal activities of chitinase and chitobiase in the digestive tract and serum of cod, Gadus morhua (L.)

No statistically significant seasonal variations of chitinase and chitobiase in cod, Gadus morhua , stomach, intestine or serum could be detected. Enzyme activity often varied considerably (up to six-fold) between fish captured at the same time, but no correlation could be detected between the presence of chitin or the amount of prey in the stomach and the activities of chitinase and chitobiase. Lysozyme was absent from all the tissues tested.     

Effective production of N-acetyl-β-D-glucosamine bySerratia marcescens using chitinaceous waste

The strain ofSerratia marcescens QM B1466 produces selectively large amount of chitinolytic enzymes (about 1mg/L medium). Enzymatic hydrolysis of chitin to N-acetyl-β-D-glucosamine (NAG) was performed with a system consisting of two hydrolases (chitinase and chitobiase) produced by optimization of a microbial host consuming chitin particles. For the development of Large-scale biological process for the production of NAG from chitinaceous waste, the selection and optimization of a microbial host, particle size of chitin and pretreatment of chitin source were investigated. Also, the effect of crab/shrimp chitin sources and initial induction time using chitin as a sole carbon source on chitinase/chitobiase production and NAG production were examined. Crab-shell chitin(1.5%) treated by dilute acid and, ball-milled with a nominal diameter less than 250m gave the highest chitinase activity over a 7 days culture. Crude chitinase/chitobiase solution obtained in a 10 L fed-batch fermentation showed a maximum activities of 23.6 U/mL and 5.1 U/mL, respectively with a feeding time of 3 hrs, near pH 8.5 at 30°C.     

Chitinolytic enzymes and the bacterial microflora in the digestive tract of cod, Gadus morhua

The levels of chitinolytic enzymes and chitinolytic bacteria in the digestive tract of feeding and fasting cod were compared. Enzyme activities within a given tissue were of a similar order irrespective of the presence of chitinolytic bacteria and/or chitin. Cod chitinase and chitobiase are therefore endogenous and constitutive enzymes. Fasting cod had similar numbers of bacteria within the gastro-intestinal compartments as feeding fish. Representative bacterial isolates from fasting fish were characterized.     

Chitinolytic activities in the gut of Aedes aegypti (Diptera:Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut.     

Modelling gastric evacuation in gadoids feeding on crustaceans

A mechanistic, prey surface‐dependent model was expanded to describe the course and rate of gastric evacuation in predatory fishes feeding on crustacean prey with robust exoskeletons. This was accomplished by adding a layer of higher resistance to the digestive processes outside the inner softer parts of a prey cylinder abstraction and splitting up the prey evacuation into two stages: an initial stage where the exoskeleton is cracked and a second where the prey remains are digested and evacuated. The model was parameterized for crustaceans with different levels of armour fed to Atlantic cod Gadus morhua or whiting Merlangius merlangus and recovered from the stomachs at different post‐prandial times. The prey species were krill Meganyctiphanes norvegica; shrimps and prawns Crangon crangon, Pandalus borealis, Pandalus montagui and Eualus macilentus; crabs Liocarcinus depurator and Chionoecetes opilio. In accordance with the apparent intraspecific isometric relationship between exoskeleton mass and total body mass, the model described stage duration and rate of evacuation of the crustacean prey independently of meal and prey sizes. The duration of the first stage increased (0–33 h) and the evacuation rate of both stages decreased (by a half) with increasing level of the crustacean armament in terms of chitin and ash. A common, interspecific parameterization of the model within each of the categories krill, shrimp and crab can probably be used if the contents of chitin and ash are similar among prey species per prey category. The model offers a simple way for estimating evacuation rates from stomach content data in order to obtain food consumption rates of wild fishes, provided that information about digestion stage of crustacean prey is available.     

Dietary preference and digestive enzyme activities as indicators of trophic resource utilization by six species of crab

The digestive physiology and stomach contents of six crab species from a variety of habitats were investigated to provide an indication of their digestive capability and dietary preferences. Stomach contents varied between species, but the key enzymes present were generally consistent with the types of dietary material being ingested. Nectocarcinus integrifons (red rock crab) consumed large quantities of seagrass and had high cellulase activity (0.02+/-0.004 units mg-1) to digest the constituent cellulose. Petrolisthes elongatus (porcelain crab) ingested brown and green phytoplankton and algae and had considerable laminarinase (0.35+/-0.08 units mg-1) and beta-glucosidase (0.025+/-0.005 units mg-1) activities to digest the laminarin in its diet. Leptograpsus variegatus (omnivorous swift-footed shore crab) had high activities of protease (1.2+/-0.02 units mg-1), alpha-glucosidase, and alpha-amylase and appeared well equipped to utilize both dietary protein and carbohydrate. Stomach contents in Nectocarcinus tuberculosus (velvet crab) and Carcinus maenas (green crab) also suggest that these species are omnivorous. N. tuberculosus had high cellulase and chitinase for digesting the cellulose in plants and the chitin in invertebrate shells respectively. C. maenas had intermediate digestive enzyme levels and may employ more of a generalist feeding strategy than other species. Plagusia chabrus (speedy crab) is carnivorous, consuming encrusting bryozoans, hydroids, crustaceans, and fish. It has high protease activity, particularly trypsin (0.73+/-0.12 units mg-1), to digest the protein in its animal prey. Each species of crab studied had a complex suite of digestive enzymes, the relative activities of which reflected individual and very different species-specific dietary niches.     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.     

A quantitative study of some digestive enzymes in the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer

Digestive enzyme distribution and activity in the digestive tracts of the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer were studied. Quantitative determinations of digestive enzymes in the guts of both fishes showed that they were capable of digesting carbohydrates and proteins in their diet. The carbohydrases, amylase, laminarinase, maltase, sucrase and trehalase were detected in the rabbitfish; their activities being mainly in the stomach, intestine and pyloriccaeca. Amylase, maltase, trehalase and chitinase activities were recorded in the gut of the sea bass, primarily in the intestine and the pyloriccaeca. Their activities were significantly lower than those in the rabbitfish. Proteases (pepsin, chymotrypsin, elastase, leucine aminopeptidase and trypsin) were found in both the rabbitfish and the sea bass. Pepsin activity however, was higher in the sea bass; while trypsin and chymotrypsin activities were higher in the rabbitfish. The activities of the various digestive enzymes in both fishes are discussed in relation to their feeding habits.     

Chitinase-overproducing mutant of Serratia marcescens.

Genetic modification of Serratia marcescens QMB1466 was undertaken to isolated mutants which produce increased levels of chitinolytic activity. After mutagenesis with ultraviolet light, ethyl methane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, 19,940 colonies were screened for production of enlarged zones of clearing (indicative of chitinase activity) on chitin-containing agar plates. Forty-four chitinase high producers were tested further in shake flask cultures. Mutant IMR-1E1 was isolated which, depending on medium composition, produced two to three times more than the wild type of the other components of the chitinolytic enzyme system--a factor involved in the hydrolysis of crystalline chitin and chitobiase. After induction by chitin, endochitinase and chitobiase activity appeared at similar times for both IMR-1E1 and QMB1466, suggesting possible coordinate control of these enzymes. The results are consistent with IMR-1E1 containing a regulatory mutation which increased production of the components of the chitinolytic enzyme system and/or with IMR-1E1 containing a tandem duplication of the chitinase genes. The high rate of reversion of IMR-1E1 to decreased levels of chitinase production suggests that the overproduction of chitinase by IMR-1E1 is due to a tandem gene duplication.     

Crystallization of recombinant chitobiase from Serratia marcescens.

We are currently investigating the biochemical and structural properties of both chitin degrading enzymes chitinase and chitobiase from Serratia marcescens. Previously we have reported the first crystallization and characterization of chitinase crystals (Vorgias et al., 1992). In this communication we present the first crystallization of chitobiase. The protein was synthesized in Escherichia coli and purified to homogeneity using cation exchange chromatography and fast protein liquid chromatography. The crystals have the shape of small prisms and the space group is P2(1) with beta = 101.0 degrees and unit cell dimensions a = 63.2 A, b = 133.2 A, c = 55.1 A. They diffract X-rays to about 2.5 A resolution and are suitable for three-dimensional structural analysis.     

Role ofCristispira sp. and other bacteria in the chitinase and chitobiase activities of the crystalline style ofCrassostrea virginica (Gmelin)

Activity was found for chitinase and chitobiase in the crystalline styles of American oysters (Crassostrea virginica Gmelin) collected from the Chesapeake Bay (Maryland, USA). The oysters were maintained in tanks on natural food from a constant flow of unfiltered estuarine water. Chitinase and chitobiase specific activities were compared with total, viable, and chitinoclastic bacterial counts andCristispira counts. Regression analyses revealed that one correlation, chitobiase vsCristispira, was significant (P < 0.05). Several oysters were fed chitin in the presence or absence of chloramphenicol. Although no chitinoclasts were present in the antibiotic-treated oysters, the treatment means did not differ significantly (P > 0.05) for either chitinase or chitobiase activity. In several cases with both chitin-fed and naturally fed oysters, enzyme activity was found when noCristispira were present. The results of the investigations suggest that the oyster produces chitinase and chitobiase endogenously.     

COMPARATIVE FEEDING ECOLOGY OF SPINNER DOLPHINS (STENELLA LONGIROSTRIS) AND FRASER'S DOLPHINS (LAGENODELPHIS HOSEI) IN THE SULU SEA

Fraser's and spinner dolphins are known to feed on mesopelagic prey, bur their diets and feeding ecologies have not been compared in areas where they are observed together. In this study we examined the stomach contents of both species caught incidentally in a driftnet fishery for tuna in the eastern Sulu Sea. Importance of prey items was determined using the percent occurrence, percent number, and volumetric methods. Mesopelagic fishes, particularly myctophids (mainly Ceratoscopelus warmingi, Diaphus spp. and Myctophum asperum ), were the most important component in the diet of spinner dolphins, whereas in Eraser's dolphins, mesopelagic cephalopods ( Abraliopsis, Onychoteuthis, Histioteuthis , and Chiroteuthis ), and crustaceans ( Notostomos elegans, Acanthephyra quadrispinosa , and Acanthephyra carinata ) appeared to be equally important as the myctophid species. Fraser's dolphins appeared to feed preferentially on larger prey and had more diversified prey (Shannon's diversity index = 1.2) than spinner dolphins (diversity index = 0.9). Vertical distributions of the prey items summarized from published literature indicate that spinner dolphins forage in the upper 200 m and probably occasionally to as deep as 400 m, whereas Fraser's dolphins have a wider vertical foraging range, from near the surface to probably as deep as 600 m.     

Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol

The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents.     

Insect resistance of transgenic tobacco expressing an insect chitinase gene

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control     

Maintaining fish diversity in Thailand: variations in foraging behavior

We examined foraging adaptations and diet among dominant fishes within two species-rich riffle habitats in Thailand headwater rivers during the early and late portions of the dry season. All species exhibited diel temporal variations in feeding activity. Some species fed principally during daylight, while others fed mainly during darkness. Feeding patterns within species were generally spatially and temporally consistent. All seven fishes exploited the same benthic prey, but their foraging activities were related to spatially distinct water velocities. Diets overlapped with Ephemeroptera and Trichoptera the quantitatively most important prey. Ostracods, while not a large contributor to diet biomass, were numerically important in the diet of Homaloptera smithi. Trophic diversification through diel temporal feeding patterns and microhabitat separation by water velocity are major tactics in maintaining high diversity in riffle assemblages in Thai headwater rivers.     

Comparison of the chitinolytic properties of Clostridium sp. strain 9.1 and a chitin-degrading bacterium from the intestinal tract of the plaice, Pleuronectes platessa (L.)

The chitinolytic properties of a facultatively anaerobic bacterium isolated from the hindgut of plaice were compared with those of Clostridium sp. strain 9.1, a bacterium isolated from anoxic estuarine sediment. The chitinolytic enzyme systems of the gut isolate and strain 9.1 both released N,N'-diacetylchitobiose (NAG2) as the major hydrolysis end-product. During the hydrolysis of chitin, there was transient accumulation of a non-sedimentary chitin fraction which was not detectable by high-performance liquid chromatography. Growth on NAG2 repressed chitinase synthesis in the gut isolate but not in the Clostridium species. Thiol reagents were strongly inhibitory to the chitinase of the strict anaerobe but did not affect the hydrolytic enzymes of the gut isolate. When the two bacteria were cocultured with chitin as the sole carbon and energy source, Clostridium sp. strain 9.1 was always outcompeted. Experiments with batch and phauxostat cultures showed that the competitiveness of strain 9.1 could be improved dramatically by the inclusion in the cocultures of a non-chitinolytic bacterium capable of fermenting chitin oligomers. The cooperation between the oligomer-fermenting species and the Clostridium sp. is discussed in relation to the regulation of chitinolytic activity in the latter organism.     

Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii

Chitinase activity was measured in extracts of mycelial cells of Mucor rouxii as a function of the culture age. There was a peak of specific activity at the mid-exponential phase of growth (10 h), which paralleled chitin synthase activity. An additional peak of chitinase with higher specific activity was detected in 4 h cultures, which coincided with the onset of germination. Purification of chitinase activities from the cytoplasm revealed two enzymes, I and II, with different molecular mass and ionic charge. Antibodies induced with chitinase I did not cross-react with chitinase II. Both enzymes digested nascent chitin preferentially over preformed chitin, yielding diacetylchitobiose as the sole product of hydrolysis.     

Seasonal change in the gastric evacuation rate of rainbow trout feeding on natural prey

Seasonal changes in the gastric evacuation rate ( R ) and gut contents of a wild population of rainbow trout Oncorhynchus mykiss feeding on natural prey at four water temperatures (2, 7, 9 and 12° C) were measured. The R and mass of the gut contents increased with water temperature, and prey items changed seasonally. These results suggest that the R of fish feeding on natural food depends primarily on water temperature, with their consumed prey being a secondary factor.