Incidence and estimation of chitinase activity associated with marine fish and other estuarine samples

A procedure for the determination of chitinase activity was adapted for the seawater environment. Preliminary data indicate that the controlled bacterial environment within the digestive tracts of marine fishes and possibly other marine animals plays a significant role in the decomposition and recycling of chitin. It is estimated in the stomachs of a single population of Enophrys bison (buffalo sculpin) of 1×10⁵ fish that ca. 16 metric tons of chitin could be decomposed annually.Published as Technical Paper No. 4434, Oregon Agricultural Experiment Station. This research was supported by NIH training grant 5TO1 GM-00704 and NSF grant DES73-06611 AO2.     

Role of bacteria with regard to chitin degradation in the digestive tract of the cod Gadus morhua

Numbers of aerobic, heterotrophic and chitinolytic bacteria in stomach, pyloric caeca and intestine of eight specimens of freshly caught cod (Gadus morhua L.) were determined by the spread plate method (medium: sea water agar, including 1% chitin). In crude enzyme extracts, chitinase activity was assayed according to the end product measurement. Results revealed no correlation between the number of chitinoclasts and the level of chitinase activity in any part of the digestive tract of cod. In another series of studies, antibiotics (Ampicillin/Sisomicin, or Chloramphenicol) were applied via tank water and feed. In most cases, antibiotic treatment resulted in the complete elimination of bacteria from the digestive tract. In contrast, up to 1.0×10⁹ chitinoclasts per g wet wt were determined in control fish. Chitinase did not reflect the absence or presence of bacteria: no significant difference in enzyme activities of cod treated with Ampicillin/Sisomicin and non-treated fish could be demonstrated. A decrease of chitinase activities of cod exposed to Chloramphenicol is interpreted as a toxic effect of this antibiotic on the fish. It is concluded that chitinase activities measured in the digestive tract of G. morhua primarily originate from fish tissues.Results presented in this paper are part of the author's Ph.D. dissertation. Authorization for pre-publication was given by the Graduation Committee of the Biology Department, University of Hamburg     

Isolation of cellulolytic microbes from the intestinal tract of the pinfish,Lagodon rhomboides: size-related changes in diet and microbial abundance

Pinfish, Lagodon rhomboides (Linneaus), undergo a gradual ontogenetic dietary shift during their first year of life, marked by an increase in the consumption of plant material. To determine if this shift in diet was associated with a change in the microbial flora of the intestinal tract that may assist in degradation of plant material, stomach contents were analyzed and microbes in the intestinal tract were isolated from fish ranging from 20 to 139 mm standard length. These fish were collected from Core Sound, North Carolina, USA between March and September 1991. Plant material increased from 16% of dry weight of stomach contents in pinfish under 40 mm standard length (SL) to 65% in pinfish above 120 mm SL, confirming previous observations of a diet-related ontogenetic change in L. rhomboides. Comparison of the total cultivatable facultative and anaerobic microbial flora isolated from the intestinal tract contents of pinfish ranging in size from 26 to 139 mm SL showed a 10-fold increase between fish <40 and fish >40 mm SL, with maximum population densities of approximately 2x10⁷ colony forming units (CFU) g^-1 of intestine including contents. The percentage of microbial isolates examined capable of hydrolyzing carboxymethylcellulose (CMC) increased from 12% in fish <40 mm SL to 13 to 50% in fish >40 mm SL, although there was no strict increase with increasing fish size classes. Although the percentage of CMC-hydrolytic microbial isolates varied with respect to fish SL, the percentage of skim-milk hydrolytic (proteolytic) isolates remained relatively constant (4% of total isolates) irrespective of fish SL and dietary composition. Results presented in this study document the first isolation of carboxymethylcellulase producing microbes from the intestinal tract of any fish and demonstrate that the ontogenetic dietary shift in L. rhomboides is paralleled by qualitative and quantitative changes in the intestinal microbial community. The use of strict anaerobic sampling methods in the preparation of intestinal contents from wild-captured fresh specimens was essential in obtaining these isolates.     

Influence of bacteria on growth and hemolysin production by the marine dinoflagellate Amphidinium carterae

The effect of various bacteria on the growth of and hemolysin production by Amphidinium carterae was studied. The algal culture had an indigenous bacterial flora of Moraxella and Pseudomonas and these could not be eliminated by treatment with bactericidal antibiotics like gentamicin. Various bacteria like Micrococcus, Aeromonas, Vibrio, and Moraxella-like bacteria were added to A. carterae cultures to study their effect on growth and hemolysin production. Micrococcus and Aeromonas were found to improve the growth marginally. Hemolysin titres were considerably higher in A. carterae cultures supplemented with bacteria. Received: 27 May 1997 / Accepted: 22 July 1997     

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.     

Possible role of marine bacteria in providing the creosote-resistance of Limnoria tripunctata

Electron microscopic examination of thin-sectioned Limnoria tripunctata from creosoted and untreated wood substrates from Panama, Florida, and laboratory aquaria, coupled with limnorian behavioral studies revealed that bacteria may contribute to the wood boring, nutrition, and creosote-resistance of these isopods. Ingested along with wood fragments and encased in a peritrophic membrane in the isopod intestine, these bacteria, upon lysis, may provide supplemental nutrition to the nitrogen-poor wood diet of L. tripunctata. Ingested material has never been observed in the digestive diverticula of limnorians and the presence of the peritrophic membrane in the isopod intestine has been correlated with feeding. Isopods from creosoted wood differed from those inhabiting untreated wood in that the former contained relatively larger and more diverse bacterial populations both on their exoskeletons and in their gut contents. These isopods harbored bacteria, which apparently bypassed the peritrophic membrane and lived in association with the isopod intestinal lining. These gut-associated bacteria were lost when laboratory isopods reared on creosoted wood were transferred to untreated wood. Laboratory isopods reared solely on creosoted or untreated wood were exposed to creosoted and untreated wood substrates, both sterilized and unsterilized. Boring and mortality data from these studies indicated that the creosote-reared population had a microbial flora whose activity facilitated isopod colonization of creosoted substrates. We present the hypothesis that creosote hydrocarbons provide nutrition for isopod-associated bacterial populations and that L. tripunctata may benefit from a concomitant bacterial detoxification of the creosote.     

Antimicrobial activity of extracts of Caribbean gorgonian corals

Extracts of 39 species of Caribbean gorgonians were tested for antimicrobial activity against 15 strains of marine bacteria. The bacteria consisted of three opportunistic pathogens, Vibrio parahaemolyticus, Leucothrix mucor, and Aerococcus viridans, and 12 strains isolated from either healthy or decayed gorgonians. Overall, only 15% (79 out of 544) of the tests resulted in antibacterial activity with 33% (13 out of 39) of the gorgonians inhibiting only one bacterial strain and 23% (9 out of 39) showing no activity. The extracts of four Pseudopterogorgia species showed relatively high levels of activity, inhibiting 43 to 86% of the bacterial strains. The potency of the active Pseudopterogorgia species was variable, however, and three additional Pseudopterogorgia species were inactive against all bacterial strains. With the exception of one sensitive strain, Vibrio species were resistant to gorgonian metabolites. Our results indicate that organic extracts of most Caribbean gorgonians do not possess potent, broad-spectrum antibacterial activity inhibitory to the growth of opportunistic marine pathogens and bacteria associated with healthy and decayed gorgonian surfaces. These findings suggest that the inhibition of bacterial growth is not the primary ecological function of gorgonian secondary metabolites and that bacteria may not be important selective agents in the evolution of gorgonian secondary chemistry.     

Heterotrophic bacteria in Long Island Sound

Correlated with a study of heterotrophic bacteria from cultures of larval bivalve molluses, bacteria were studied from the water column over an oyster bed in Lont Island Sound, USA. Samples taken biweekly for 1 year showed bacterial densities ranging from 1.2x10³/ml in summer to 3.8x10⁴/ml in winter. Variation in seasonal density mirrored that of phytoplankton. Gram-negativ rods constituted 99% of the isolate. Eight genera were identified, but 92.3% of these were Achromobacter, Flavobacterium, and Pseudomonas. Pseudomonads dominated the summer flora, whereas flavobacteria were most abundant in spring. Types varied seasonally, but proteolytic, lipolytic, and amylolytic activity appeared independent of season.     

Larval settlement in the serpulid polychaete Hydroides elegans in response to bacterial films: an investigation of the nature of putative larval settlement cue

Larval settlement in the marine polychaete Hydroides elegans (Haswell) is induced by certain bacteria in marine biofilms. The exact nature of the settlement cue that larvae of H. elegans receive from these bacteria remains unknown. In this study, we revealed some properties of the bacterially derived larval settlement cue by investigating the larval settlement inductive activity of two bacterial strains after various treatments. These two bacterial strains, Roseobacter sp. and an α-subclass Proteobacteria, are highly inductive to larval settlement of H. elegans. The larvae responded similarly to Roseobacter and Proteobacteria in all the larval settlement bioassays, suggesting that the larval settlement-inducing substances produced by these bacteria may share common characteristics. First of all, the larvae did not settle in the seawater conditioned by the bacteria attached as a film or by the bacteria that were freely suspended in seawater. The results suggest that the putative larval settlement cue is not released into seawater and, therefore, should be associated with the surface of the bacteria. Secondly, formaldehyde treatment entirely eliminated the larval settlement induction activity of the bacterial films, and streptomycin treatment reduced the percentage of larval settlement on the bacterial films in a concentration-dependent manner. Since both treatments can kill bacteria with little damage to the surface chemistry of bacterial cells, the decline in larval settlement is suggested be due to a reduction of the viable bacterial population in the bacterial films. In fact, the reduction of larval settlement in the streptomycin treatments coincided with the decrease in viable bacterial populations in broth cultures containing respective concentrations of streptomycin. These results suggest that the viability of Roseobacter and Proteobacteria is important to their settlement induction effect. Since the larval settlement induction activity of the bacterial strains appears to correlate with their viability, we suggest that the putative larval settlement cue is derived from a metabolic pathway in the bacteria and that the cue is exported to and concentrated at the extracellular polymer matrix of the bacterial cell, at which the larvae establish contact with the bacteria. The larval settlement cue may be highly susceptible to degradation so that a metabolically active bacterial film is needed to maintain the putative cue at a concentration that surpasses the threshold for induction of larval settlement. Received: 14 October 1998 / Accepted: 5 September 2000     

Modified dehydrogenase enzyme assay for evaluation of the influence of Hg,Cd, and Zn on the bacterial community structure of a wastewater treatment plant

In this study, a traditional assay was modified to evaluate the effect of Hg, Cd, and Zn on the bacterial community of a sequencing batch reactor and activated sludge plants and heavy metal-resistant bacterial species were determined. After the isolation of metal-resistant bacteria, their 16S rRNA gene fragments were sequenced. The BLAST program was used to compare the resulting 16S rRNA sequences with those in GenBank database to identify the isolated bacterial species. Hg was found to be the most toxic metal for both bacterial communities investigated. Sequence batch reactor bacteria were comparatively more resistant to Hg, Cd, and Zn than those from activated sludge. The resistant strains were close to the members of genus Pseudomonas, Kocuria, Stenotrophomonas, Enterococcus, and Staphylococcus. The modified dehydrogenase enzyme assay seems to be simple, robust, and competent for evaluation of the impact of metals on bacterial activity. Sequencing batch reactor systems should be preferred over activated sludge when wastewaters containing hazardous metals are to be treated.     

Evidence for n-alkane consumption and oxidation by filamentous cyanobacteria from oil-contaminated coasts of the Arabian Gulf

Microcoleus chthonoplastes and Phormidium corium were isolated from microbial mats covering all sediments along the Arabian Gulf coasts. These isolates could consume and oxidise n-alkanes. The establishment of axenic cultures faced the problem that with progressive axenity the cyanobacterial growth seemed to cease. The associated organotrophic bacteria, Rhodococcus rhodochrous, Arthrobacter nicotianae, Pseudomonas sp. and Bacillus sp., could utilize n-alkanes. The total number of these organotrophs was about 2×10⁶ cells g⁻¹ fresh culture, and R. rhodochrous was the most dominant. In order to test the potential of cyanobacteria for n-alkane consumption, experiments were constructed to rule out the role of the associated organotrophic bacteria. Aliquots, 0.5 g fresh cyanobacterial samples, each containing about 1×10⁶ organotrophic bacterial cells (≡0.001 mg fresh bacteria) were incubated in inorganic medium aliquots supplied with an n-alkane. The same was repeated using 1.0×10⁶ cells each of the four organotrophic bacteria instead of the cyanobacterial samples. The nonaxenic cyanobacterial samples consumed up to 60% of the available alkane, whereas no detectable consumption was measured in any of the pure organotrophic bacterial cultures. For all organotrophic bacteria, the numbers had to be increased ten-thousand times in order that detectable alkane consumption might become measurable. The fatty acids resulting from the n-alkane oxidation were found incorporated in cell lipid classes characteristic of cyanobacteria, namely in galactolipids and sulfolipids. These results may imply that the two test cyanobacteria contribute directly to n- alkane uptake and oxidation. Received: 6 May 1997 / Accepted: 2 October 1997     

Bacterial colonization of the phyllosphere of nineteen plant species and antimicrobial activity of their leaf secondary metabolites against leaf associated bacteria

Summary. The scope of this work was to examine whether leaf constitutive secondary metabolites play a role in determining bacterial colonization of the phyllosphere. To this aim, we surveyed nineteen native or cultivated plant species that share a common bacterial pool in a North Mediterranean area, and estimated the size of total and ice nucleation active (INA) bacterial populations on their leaves. Large differences in the colonization of their phyllosphere were found; the population size of epiphytic bacteria ranged from 7.5 × 10² to 1 × 10⁶ CFU/g fresh weight, in eucalypt and celery, respectively. Species native in Mediterranean-type climate areas, particularly those belonging to the group of aromatic plants, are characterized by scarce presence of INA bacteria. The antibacterial activity of essential oils, surface phenolics and leaf tissue extracts was also estimated against the INA strains P. syringae and E. herbicola, isolated from two of these plant species. E. herbicola proved more sensitive than P. syringae. Of the species examined, oregano [Origanum vulgare L. subsp. hirtum (Link.) Ietswaart], an aromatic plant, had the highest antimicrobial activity, whereas six species showed no activity at all. Further experiments were performed with oregano and bean (Phaseolus vulgaris L.) that represent two extremes in their secondary metabolite content. Both plants were inoculated with P. syringae. By the end of incubation, the bacterial population on bean plants was about 100 times higher than that on oregano leaves. Scanning electron micrographs showed that bacterial growth on oregano leaves was confined to sites away from glandular hairs. Results from the bacterial colonization survey together with those from the toxicity tests showed that all species rich in antibacterial secondary metabolites harbored low leaf bacterial populations. These results provide substantial evidence that leaf secondary metabolites function as constitutive defense chemicals against microbial invasions. However, the fact that species with non- or moderately active leaf secondary metabolites are not always highly colonized suggests mediation of other unknown factors, the contribution of which requires further investigation.     

Distribution de certaines bactéries hétérotrophes aérobies dans les sédiments profonds de Méditerranée Orientale

Ninety five sediment samples were extracted from 16 cores collected at depths from 283 to 4,350 m in the Eastern Mediterranean Sea. Generally, the microbial population of superficial layers is lower than 10⁵ bacteria/g sediment. At depths below 1,000 m this population is lower than 10⁴. Bacterial counts at different levels of the sediment show that the microbial population is only important in the superficial layer. Below the surface, bacterial counts are generally between 10² to 10³ cells/ml. Some levels appear to be sterile; none of the 12 culture media gave rise to development with the sediment samples. Studies of some physiological groups of bacteria show scarcity of chitinoclastic and agarolytic bacteria and absence of cellulolytic and sulphate reducers. On the other hand, amylolytic, gelatinolytic, lypolytic and nitrate-reducing bacteria are widely distributed among the microbial population of marine sediments. This population of deep sediments includes a few auxotrophic bacteria.     

Characterizing the resident, fermentative microbial consortium in the hindgut of the temperate-zone herbivorous fish, Hermosilla azurea (Teleostei: Kyphosidae)

The zebraperch, Hermosilla azurea Jenkins and Evermann, a warm-temperate marine fish species with a strictly macroalgal diet, has a relatively long digestive tract with an enlarged hindgut and an associated blind caecum (HC). In zebraperch sampled off Santa Catalina Island, California (33°19′42′′N; 118°18′37′′W) in years 1995 through 2001, direct cell counts, gut epithelium assessment of bacterial attachment, and short-chain fatty acid (SCFA) analyses verified that the zebraperch HC possesses a dense and morphologically diverse, fermentative microbiota. Bacterial cell counts and morphological diversity were significantly higher in HC contents compared to anterior gut regions, suggesting that microbial populations were growing along the digestive tract. Similarly, electron micrographs of the HC epithelium revealed attached microbes, further supporting the possibility that these organisms constitute resident microbiota. Five different SCFAs were detected in all three regions of the digestive tract, but levels were up to three times greater in HC contents. Acetate was consistently the prevailing SCFA in all gut regions. Sequence analysis of bacterial 16S rDNA was used to identify predominant bacterial groups in HC contents. Of the seven main bacterial types identified, Enterovibrio spp. were the dominant bacteria in HC contents followed by species of Bacteroides,Faecalibacterium, and Desulfovibrio. Taken together, our findings show that the zebraperch HC harbors a consortium of microbes that appears to assist in the breakdown of algal polysaccharides in the herbivorous diet of the fish.     

Stomach contents and size-frequency distributions of two coexisting sea star species,Astropecten aranciacus and A. bispinosus,with reference to competition

Stomach contents of the sea star species Astropecten aranciacus (L.) and A. bispinosus (Otto) consisted of roughly the same sort of prey, mostly bivalves and irregular sea urchins. However, the two predators ingested prey of different size classes. The stomach contents of A. bispinosus reflected the size distribution of the benthic macrofauna, while larger prey was more frequent in the stomach contents of A. aranciacus. As the density of prey in the investigated area is low, A. aranciacus and A. bispinosus are likely to compete for food. Possible mechanisms reducing the effect of competition are discussed.     

Food of Anguilla anguilla in Lake Manzalah,Egypt

The food and feeding habits of Anguilla anguilla L. in Lake Manzalah (Egypt) have been studied. Analysis of stomach contents revealed that eels alter their type of diet as they grow. Their feeding rate varies also with season, size and environmental temperature. A negative correlation was found between length and feeding index of A. anguilla.     

铜胁迫对农田土壤酶活性、细菌和古菌数量的影响

为了研究铜矿周边农田土壤中不同Cu含量对土壤酶活性、细菌和古菌基因拷贝数的影响,在铜矿周边农田土壤中采集了8个不同Cu含量的土壤,测定土壤的酶活性、细菌和古菌基因拷贝数,结果表明,土壤总Cu含量为28.30~1 019.27mg·kg~(-1),酸可提取态Cu含量为335~415.11 mg·kg~(-1),细菌16S rRNA基因拷贝数在每克干土3.10×10~(10)~1.84×10~(11)个之间,古茵16S rRNA基因拷贝数在每克干土737×10~8~6.82×10~9个之间。土壤总Cu和酸可提取态Cu含量对土壤脲酶活性影响较大,但对土壤脱氢酶和转化酶活性影响不显著。土壤细菌基因拷贝数均与总Cu含量、各形态Cu含量呈极显著的负相关关系,同样土壤古菌基因拷贝数与总Cu含量、各形态Cu含量也均呈极显著的负相关关系,说明Cu胁迫对土壤细菌和古菌活性具有较大的抑制作用。     

Inhibition of attachment of larval barnacles,Balanus amphitrite,by bacterial surface films

Films of bacteria on solid substrata can positively or negatively influence the attachment of marine invertebrate larvae. Effects of marine bacteria on the attachment of cypris larvae of the barnacle Balanus amphitrite Darwin were examined in the laboratory. Bacteria, grown to mid-exponential phase and allowed to adsorb irreversibly to polystyrene petri dishes, attached in densities of 10⁷ cells cm^-2. Assays (22h) were used to compare the effects of adsorbed cells of 18 different bacterial species on larval barnacle attachment. Most of the adsorbed bacteria either inhibited or had no effect on larval attachment compared to clean surfaces. Experiments testing the effect of larval age on barnacle attachment were conducted with six species of bacteria and showed that older larvae attached in higher percentages to clean surfaces and that bacterial films generally inhibited larval attaschment. Both the species of bacteria and the in situ age of the adsorbed bacteria affected barnacle attachment response: older films of Deleya (Pseudomonas) marina were more inhibitory. Bacterial extracellular materials may be involved in the inhibitory process.     

The enumeration and characterization of bacteria and fungi associated with marine wood-boring isopods,and the ability of these microorganisms to digest cellulose and wood

Bacteria and fungi associated with the marine wood-boring isopods Limnoria lignorum and Sphaeroma serratum, and with their wood burrows and seawater were investigated. Plate counts using nutrient agar and Czapex Dox agar media in natural and in artificial seawater were used. The diversity and number of bacteria associated with the two isopods were much higher than those associated with wood or seawater, whereas the opposite was true for fungi. The hydrolytic activity of the predominant genera on carboxymethylcellulose and on different types of wood was investigated. The magnitude of fungal cellulase activity exceeded that of bacteria. Activities of bacterial extracellulases exceeded those of intracellulases, while the opposite was observed for fungi. Cellulolytic activity of the predominant isolates biodegraded labkh, peach, white oak and mahogany in decreasing order of activity. Evidence provided by the cellulolytic activities, detected in woodcontaining cultures, suggests that the predominant micro-organisms isolated may play an important role in the woodboring process. Specimens used in the present study were collected from Port Fouad at Port Said Harbour, Egypt.     

Lysozyme and chitinase in blood and lymphomyeloid tissues of marine fish

The enzyme lysozyme (muramidase) has a degrading effect on chitin, but chitinase is more effective in this respect. Both enzymes occur in fish, but their distribution and function are poorly known. Activities of lysozyme and chitinase were measured in body fluids and tissues of marine fish. Lysozyme was estimated by a plate method, chitinase by a viscosimetric method with glycol chitin as substrate. pH optima and other characteristics were studied. Lysozyme was found in lymphomyeloid (haematopoietic) tissues of chondrichtyan fish (rays, shark, Chimaera monstrosa) and in the plasma and lymph of certain teleosts. Chitinase occurred in lymphomyeloid tissues (except thymus) of chondrichthyans and teleosts, and in the plasma and lymph of teleosts. The results indicate that these enzymes are normal components of fish blood. Probably they are produced by the granulocytes, white blood corpuscles rich in lysosomes. The enzymes might have defence functions in fish blood. Lysozyme is bacteriolytically active while chitinase possibly offers protection against chitincontaining parasites. Very high chitinase activity was found in the pancreas of C. monstrosa. The pancreatic enzyme probably has a digestive function.