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The intestinal mucosa of pike (Esox lucius L.) from the Rybinsk Reservoir served as the subject for this. Hydrolytic Activity of Symbiotic Microflora Enzymes in ...
ISSN 19950829, Inland Water Biology, 2011, Vol. 4, No. 1, pp. 72–77. © Pleiades Publishing, Ltd., 2011. Original Russian Text © G.I. Izvekova, A.O. Plotnikov, 2011, published in Biologiya Vnutrennikh Vod, No. 1, 2011, pp. 79–85.

PARASITOLOGY OF HYDROBIONTS

Hydrolytic Activity of Symbiotic Microflora Enzymes in Pike (Esox lucius L.) Intestines G. I. Izvekovaa and A. O. Plotnikovb a

Papanin Institute for the Biology of Inland Waters, Russian Academy of Sciences, Borok, 152742 Russia email: [email protected] bInstitute of Cellular and Intracellular Symbiosis, Ural Branch, Russian Academy of Sciences, ul. Pionerskaya 11, Orenburg, 460000 Russia Received April 8, 2009

Abstract—This study has revealed the existence of microflora which is, to different degrees, associated with the intestinal mucosa of pike. A total of 82 bacterial strains have been isolated. These microorganisms pro duce enzymes that hydrolyze the major food substrates (proteins and carbohydrates). These enzymes are pro duced by the pool of various microorganisms living in the intestines, as well as by separate strains isolated in pure cultures. These strains produce enzymes with different levels of activity. Most of the isolated strains (68%) produce proteases. The calculated values of the C/P factor (the ratio of carbohydrase to protease activ ity) indirectly testify to the autochthonous nature of the microflora associated with the intestinal mucosa of pike. Presumably, the contribution that the microflora enzymes make to the enzymatic activity of the pike intestine is substantial, but it is difficult to estimate now. Keywords: fish intestines, microflora, enzymatic activity. DOI: 10.1134/S1995082911010081

may be of the microbial origin. For instance, carbohy drases, chitinase, chitobiase, proteases, collagenase, and elastase that are important for sole feeding may be of bac terial origin [19]. Three types of intestinal bacteria were isolated from the gut of juvenile rohu (Labeo rohita (Hamilton)). Cultivated bacteria are able to produce extracellular protease, amylase, and cellulose, which may be important for digestion in rohu larvae [18]. It was revealed that the microorganisms isolated from the gut contents of pike (Esox lucius L.), bream (Abramis brama (L.)), roach (Rutilus rutilus (L.)), and perch (Perca fluviatilis L.) are able to produce amy lolytic and proteolytic enzymes [4]. It was shown in this study that the activities of microbial proteinases isolated from a fish gut depend on the temperature, pH, and the composition of the cultivation medium. Most of the cited papers study the microflora of the intestine contents, while the role of the microorganisms colonizing the mucous surface of the intestine remains obscure. The goal of this paper is to study the ability of bac teria associated with the pike intestinal mucosa to syn thesize proteolytic and amylolytic enzymes and assess their potential role for digestion in host.

INTRODUCTION There is a massive body of published data concerning the quantitative and qualitative composition of the enteral microflora of vertebrates, fish in particular [5, 14–16]. Most publications deal with farmed fish spe cies [12, 14]. Studies on the functions of the microflora of fish living in the wild are much less numerous. It was revealed in higher vertebrates that the microorganisms inhabiting the gut possess a specific set of enzymes that provide the hydrolysis of substrates unavailable for the substrates of the macroorganism. The participation of microflora enzymes in processes of digestion decreases the energetic and plastic expenses of an organism for the synthesis of its own digestive hydrolases [7]. It was found in three herbivorous marine fishes (Kyphosus sydneyanus (Günther), Aplodactylus arctidens (Richardson), and Odax pullus (Forster)) that amylolytic activity related to the cellular extracts of microorganisms progressively rises in the direction from the anterior to the posterior sections of the intestine, contributing in some cases one third to the total activity [21]. The enzymes of microorganisms greatly contribute to the enzymatic activity and provide energy to host [21]. Microflora pos sessing amyloytic, cellulolytic, lipolytic, and proteolytic enzymatic activities was isolated from the intestines of ten fish species [13]. The studies revealed that microor ganisms positively affect the digestive processes in fish. It is very possible that some enzymes, the activity of which was revealed in the gastrointestinal tract of Dover soles,

MATERIALS AND METHODS The intestinal mucosa of pike (Esox lucius L.) from the Rybinsk Reservoir served as the subject for this 72

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study. The gut surface was sterilized by washing with ethanol followed by dissection in sterile conditions. The washouts from the pike intestine were obtained following the modified technique of sequential enzyme desorption from the gut sections [3]: gut sec tions were placed in a flask with sterile Ringer’s solu tion (10 ml) for poikilotherm animals (pH 7.4) and shacked. These sections were sequentially transferred in sterile conditions to other flasks with Ringer’s and shacking was repeated, yielding various fractions con taining enzymes and microorganisms that are sequen tially (according to the strengths of their attachment) washed out from the mucosa surface. The first fraction D1 was obtained following 30 second shacking; other fractions D2–D5 were obtained after every further 15 min shacking. The fractions D1, D3, and D5 were used for a microbiological examination. In the sequential order of fractions from D1 to D5, i.e., as the surface is approached, the strength of the association of bacteria with the digestive–transport surfaces increases and the obtained fractions correspond to the distribution of microflora in the intestine: from the cavital (fractions D1–D2) to the deep (parietal) microflora in the fractions D3–D5. The homogenate (H) of the intestinal mucosal surface was obtained using a preliminarily washed fragment of mucosa pro cessed following the procedure described above: the fragment was grinded in a ninefold (weight to volume) amount of Ringer’s solution in the sterile mortar. The activities of hydrolytic enzymes produced by intestinal bacteria were determined both in the sam ples obtained without their separation as a pure culture and in samples of pure bacterial cultures. The activities of enzymes presented in the washouts from the intes tinal mucosa and in the mucosa homogenates were also determined. The following procedure was used to determine the hydrolytic enzyme activities without their separation in the pure culture. Either the intestinal washout or homogenate (0.1 ml) was placed in test tubes with 10 ml of liquid nutritional medium. To determine the proteolytic activity, we used dry fishpeptone broth at a concentration of 35 g (medium 1P) or 3.5 g (medium 2P) per 1 l of Ringer’s solution. To determine the amy lolytic activity, we used either the Imshenetskii medium (medium 1A) or 3.5 g of fishpeptone broth and 10 g of dissolvable starch per 1 l of Ringer’s solu tion (medium 2A). The activities of bacterial enzymes were determined following the cultivation of inoculates for three days at 25°С. Pure cultures of bacteria were produced by the inoc ulation of 0.1 ml of nondissolved washouts and those dissolved 10, 100, and 1000 times or homogenate on a solid nutritional media: “hungry” agar, blood agar, 1.5% meat–peptone agar, bismuth–sulfite agar, and Endo’s and Ploskirev’s media. After 48–72 h of cultiva tion at 25°С, the colonies were reinoculated onto slant ing nutritive agar and identified. Separated strains were identified based on morphological, tinctorial, cultural, INLAND WATER BIOLOGY

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and biochemical criteria using the plate test systems ENTEROtest and NEFERMtest (LACHEMA, Czech Republic). To assess the enzymatic activities of isolated bacterial cultures, the amount of inoculates was taken to be equal for all strains: one standard bacteriological loop (3 mm in diameter) per test tube with 10 ml of liq uid nutritional medium. The activity of hydrolytic enzymes produced by pure bacterial cultures was deter mined after 3 days of cultivation at 25°C. The strains were numbered along with the sequence of their isola tion. The ability to produce proteolytic and amylolytic activities was determined for each isolated strain. The activities of specific intestinal enzymes des orbed from the intestinal mucosa were also deter mined. The washouts of the enzymes adsorbed on the digestivetransport surface of intestinal mucosa were obtained following the same procedure as was used for studies of bacteria. The activities of intestine enzymes were determined directly in the washouts without any inoculations to media or dissolving. The amylolytic activity (AA) (sum activity of αamylase, EC 3.2.1.1; glucoamylase, EC 3.2.1.3; and the enzymes of maltases group, EC 3.2.1.20) was determined using modified Nelson’s technique by the growth of hexoses [8]. To determine proteolytic activ ity (PA) (activities of trypsin, EC 3.4.21.4; chymot ripsin, EC 3.4.21.1; and dipeptidases EC 3.4.13.1– 3.4.13.11), Anson’s technique [11] (based on an assessment of the rise in tyrosine) was applied. The intensity of developing staining proportional to the enzymatic activities was measured on a SF46 spec trophotometer. During calculations of the activities of the enzymes isolated in the pure culture of strains, the rate of substrate hydrolysis was expressed in the micro moles of reducing sugars (in the case of AA) or tyrosine (in the cases of PA) for 1 h of incubation; dur ing calculations of the enzymatic activity of bacterial assemblage without isolation in pure culture, it was expressed in micromoles of reducing sugars or tyrosine for 1h of incubation per 1 g wet weight of the gut section from which the washout was obtained. The activities of the enzymes desorbed from the intestine were calcu lated per 1 g of gut section for 1 min of incubation. RESULTS The ability to produce hydrolytic enzymes (pro tease and amylase) was determined in bacteria associ ated with intestinal mucosa following the inoculation of the intestinal mucosa washouts on nutritional media (Fig. 1). It was revealed that bacteria attached to intestinal mucosa with various strengths produce hydrolytic enzymes. Both protease and amylase activ ities of bacteria were noted in all fractions. These activities depend on the composition of the cultivation medium and decrease from fraction D1 to fraction D5. The use of such an approach made it possible to reveal the fact that an assemblage composed of various

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(а)

1 2

160

80

Д1

Д3

Д5

Д7 3

(b)

4 80

40

D1

D3

D5

D7

Fig. 1. (a) Proteolytic and (b) amylolytic activities (µmol/(g h) of enzymes of the bacteria washed out from the pike intestine. Media: (1) 1P; (2) 2P; (3) 1A; (4) 2A.

bacteria inhabiting the pike intestine is able to produce hydrolytic enzymes. The next step in the study of the trophic impor tance of microorganisms in the pike intestine was determining the ability of specific strains to produce hydrolytic enzymes. To do this, we inoculated the fractions to various solid nutrition media. In all obtained fractions, a total of 82 strains of aerobic and facultativeanaerobic heterotrophic microorganisms

were isolated. These microorganisms predominantly belong to gramnegative bacteria belonging to fam. Enterobacteriaceae, Aeromonadaceae, and Vibrion aceae [2]. It was revealed that 56 (68%) cultures manifest PA and 28 (34%) manifest AA. The ability to manifest both PA and AA was revealed in 25 (30%) cultures; the ability to manifest only PA was found in 31 (38%) cul tures; and the ability to manifest only AA was found in three (4 %) cultures. None of the studied activities was found in 23 (28%) strains. In isolated strains, the level of PA is 0.039– 0.473 μmol/h (0.162 ± 0.012 μmol/h on average). In each fraction the strains possessing either relatively high or low enzymatic activity occur (Fig. 2a). The highest average level of enzymatic activities, as well as the highest sum activity, was revealed in strains isolated from the D3 fraction; the lowest average and sum activities were found in the homogenate of the intesti nal mucosa (see table). The level of AA varies from 0.015 to 0.572 μmol/h (0.202 ± 0.028 μmol/h). Like in the strains producing proteolytic enzymes, we found strains possessing either high or low levels of amylolytic activity in each fraction (Fig. 2b). The regularities revealed in the case of bacteriaproduced amylolytic enzymes are similar to those in proteolytic enzymes: the highest average and sum activities were revealed in the D3 fraction; the lowest average and sum activities were found in the homogenate of the intestinal mucosa (see table). The values of the C/P coefficient (the proportion of the sum activity of carbohydrases of all strains to the activity of proteases) were calculated for the enzymes of bacteria washed out from the studied surfaces (see table). The classical proportions of the C/P coefficient for predatory fish (C/P < 1) were revealed in all studied fractions. The activities on the enzymes running the pro cesses of membrane digestion and desorbed from the pike intestinal mucosa were also studied (Fig. 3). The enzymatic activity spreads in all fractions quit evenly. The highest PP and AA were noted in the homogenate. Thus, this study discovered the microorganisms producing hydrolytic enzymes and associated to vari ous degrees with the pike intestinal mucosa.

(A) Average and (B) sum activities and values of the C/P coefficient of bacterial hydrolytic enzymes in various fractions from the pike intestinal mucosa, μmol/h Fraction D1 D3 D5 H

PA

AA

A

B

A

B

0.155 ± 0.026 0.199 ± 0.030 0.150 ± 0.019 0.137 ± 0.018

1.862 3.191 2.247 1.778

0.210 ± 0.056 0.231 ± 0.061 0.181 ± 0.047 0.168 ± 0.051

1.263 2.309 1.084 1.005

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(а)

0.4

49. 51. 52. 31. 50. 40. 34. 42. 44. 38. 41. 63. 65. 45. 55. 61. 56. 71. 47. 35. 66. 48. 54. 46. 72. 60. 57. 62 2. 15. 77. 16. 18. 27. 9. 25. 1. 17. 67. 10. 8. 68. 26. 5. 20. 23. 21. 12. 28. 84. 13. 4. 70. 29. 22. 69.

0.2

D1

D3

D5

H

(b) 0.4

0.2

40. 41. 49. 52. 44. 33. 35. 54. 65. 60. 55. 46. 47. 45. 37. 66. 15. 25. 16. 8. 67. 11. 23. 28. 29. 84. 13. 69.

D1

D3

D5

H

Fig. 2. (a) Proteolytic and (b) amylolytic activities (µmol/h) of enzymes of the bacterial cultures from the pike intestine. The numbers of the strains are given on the abscissa.

DISCUSSION The list of important functions of the bacteria inhabiting fish intestines includes the participation in the degradation of complex molecules such as starch, cellulose, phospholipids, chitin, and collagen, which indicates that microflora makes a contribution to fish digestion [12]. Finding hydrolytic activity in many isolated strains suggests the possible participation of these microbes in the hydrolysis of the main food substrates’: proteins and carbohydrates entering the pike intestine. Since pike is a typical predator in regards to its mode of feed ing, it is not surprising that bacteria producing pro teolytic enzymes prevail in its intestine. In herbivorous fish one of the main functions of intestinal microflora is the decomposition of carbohydrates; in omnivorous fish, it is the decomposition of proteins. This is why the enzymatic activity of the digestivetract microflora is closely related to the specificity of how the fish feeds. In the intestines of herbivorous fish, carbohydrate decomposing bacteria prevail; in the zoophagous fish, proteolytic bacteria are prevalent [10]. This corre sponds to the data on the activity of various types of hydrolases of fish intestine: the activity of proteinases is higher in predatory fish than in the benthivorous and planktivorous species, where the activity of amylases is higher [9]. INLAND WATER BIOLOGY

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To assess the proportions of the activities of amy lolytic versus proteolytic enzymes in the fish intestine, the C/P coefficient is used that reflects the fish feeding type: in predators, C/P < 1; in plankti and benthivores, as a rule, C/P > 1 [9]. The C/P values revealed in this study for the bacteria washed out in different fractions μmol/(g min) 1 2

6

3

D1

D2

D3

D4

D5

D6

D7

D8

H

Fig. 3. (1) Proteolytic and (2) amylolytic activities (µmol/(g min)) of enzymes desorbed from the pike intes tinal mucosa.

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may indirectly indicate their autochthonous character; i.e., isolated bacteria are representatives not of transit but of resident microflora adapted to the host’s feeding type. The presence of bacteria producing both amy lolytic and proteolytic enzymes decreases the energy expenditure of microorganisms for the synthesis of their own enzymes and increases the availability of those components of food that do not form the basis of fish feeding (e.g., carbohydrates for predators). The results of this study correspond well with the data from other researchers concerning the ability of intestinal microflora to produce hydrolytic enzymes. For instance, >1500 strains of bacteria, of which 21.5% produced amylase, were revealed in the intes tine of seven marine species [24]. Since all the fish under study were either predators or omnivorous, it is unlikely that large amounts of starch enter the organ ism of the fish with food. However, it is obvious that amylase producers are common in the digestive tract of coastal marine fishes. It is assumed that production of amylase by bacteria plays an important role in the digestion of starch in freshwater fish. The contribution that microflora makes in the production of amylases in the digestive tract of fish may be important, but it is difficult to quantitatively establish the source of vari ous amylases [21, 23]. The production of enzymes by the symbiontic microflora was studied mainly in in vitro experiments with isolated bacterial strains. This does not allow for a direct assessment of the processes taking place in vivo. The enzymes differ in their origins. This is why attempts to distinguish intestinal enzymes, particu larly from the enzymes produced by bacteria, demand special methodical approaches. Studying the symbiontic relationship with intesti nal microflora is a complicated task due to the large number and diversity of bacteria present. In the case of mammals, the most prospective approach in studies of such complex relations seems to be the formation of simplified model systems. This may be achieved by introducing one or two members of the autochthonous flora into a sterile laboratoryraised host, which would make it possible to clarify the mechanisms of the inter relationships between the symbiont and the host, as well as between the bacteria themselves [17]. Since every strain of bacteria is specific in terms of its activ ity, studies of separate bacterial strains may character ize their roles in the digestive tract. Unfortunately, in fish, such an approach to studies of intestinal microf lora is impracticable due to impossibility of controlling fish sterility and maintaining the fish in sterile condi tions. Some researchers [6, 21] attempted to distinguish the activities of enzymes of microbial origin from the total enzymatic activity of a fish gut. If we assume that the enzymatic activity of bacteria depends on their weight, than the contribution of microbial proteinases may vary from 2 to 22% of the sum activity of the hydrolases of the enteral microbiota and fish intestinal

mucosa [6]. However, since not all microorganisms found in the gut produce proteinases and the level of activity of these enzymes differs in different strains, such an assumption seems to be quite approximate. Skea et al. [21] proposed an original method to determine the contribution that microflora enzymes in the fishgut content make to its total enzymatic activity. They homogenized and then centrifuged the gut content. In their opinion, the supernatant con tained enzymes secreted by the host and microflora. The precipitate was disintegrated by ultrasound, the fragments of microbial cells were precipitated, and the enzymatic activity of microbiota was determined in the supernatant that formed as a result of one of the centrifugations. This method seems to be prospective for studies of the contribution that the microflora enzymes make to digestive processes in macroorgan isms. However, this does not account for the potential capabilities of a bacterial cell: bacteria are able to pro duce a large quantity of exogenous enzyme in compar ison with the endogenous content. The data we obtained do not clarify the contribu tion that the microflora enzymes make to the enzy matic activity of the fish intestine; they only indicate the existence of such a contribution. It is rather diffi cult to compare the activities manifested by adsorbed and the organism’s own enterocyte membranebound enzymes on the one hand and those manifested by microflora enzymes on the other hand. The activity of intestinal enzymes is analyzed immediately after the dissection of the intestine, while the activity of the bacterial enzymes is analyzed following their cultiva tion on nutritional media for three days. This means that the conditions for determining the enzymatic activity in bacteria (compared with those during an assessment of the activities of adsorbed and, particu larly, intestinal enzymes) are more distant from the real conditions of their functioning in a living organ ism. In addition, the activity of specifically intestinal enzymes and the enzymes of microflora may be calcu lated in different ways: either per weigh of the intestine section (or of only mucosa) from which the enzymes or bacteria were washed out or per biomass of the bac teria in each fraction. Our data indicate the existence of bacteria with different levels of enzymatic activity both in the fraction that are either easy or difficult to wash out. It is possible that the microorganisms firmly attached to the mucosa make a larger contribution to the enzymatic activity of the macroorganism than bacteria which are easier to wash out and are evacu ated from the gut with peristaltic [1]. A study of the symbiontic microflora of the intesti nal tract of aquatic animals shows that the capabilities of an organism increase due to a wide spectrum of functional and biochemical activities of microorgan isms [20, 25]. INLAND WATER BIOLOGY

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CONCLUSIONS This study has revealed the existence of microflora associated to different degrees with the intestinal mucosa of pike. These microorganisms produce enzymes that hydrolyze the main food substrates’ (pro teins and carbohydrates). These enzymes are produced both by the assemblage of various microorganisms inhabiting the intestine and by separate strains. Isolated strains produce enzymes with varying activity levels. Most strains produce proteases. The calculated C/P coefficient indirectly indicates the autochthonous nature of the microflora associated with pike intestinal mucosa. Presumably, microflora enzymes contribute considerably to the enzymatic activity of the pike intes tine. However, it is difficult to assess this contribution. ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research, project no. 060448410. REFERENCES 1. Izvekova, G.I., Hydrolytic Activity of Microflora Asso ciated with Digestive–Transport Surfaces of Pike Intes tine and Triaenophorus nodulosus (Cestoda, Pseudo phyllidea) Parasitizing It, Zh. Evol. Biokhim. Fiziol., 2005, vol. 41, no. 2, pp. 146–153. 2. Izvekova, G.I., Nemtseva, N.V., and Plotnikov, A.O., Taxonomic Characteristics and Physiological Proper ties of Microorganisms from the Gut of Pike (Esox lucius), Izv. Akad. Nauk, Ser. Biol., 2008, no. 6, pp. 688–695 [Biol. Bull. (Engl. Transl.), 2008, vol. 35, no. 6, pp. 592–598]. 3. Kuz’mina, V.V., The Use of the Method of Successive Desorption of αAmylase from the Gut Segments in Studies of Membrane Digestion in Fish, Vopr. Ikhtiol., 1976, vol. 16, no. 5, pp. 944–946. 4. Kuz’mina, V.V. and Skvortsova, E.G., Bacteria of the Gastrointestinal Tract and Their Role in Digestive Pro cesses in Fish, Usp. Sovrem. Biol., 2002, vol. 122, no. 6, pp. 569–579. 5. Lubyanskene, V., Virbitskas, Yu., Yankyavichyus, K., et al., Obligatnyi simbioz mikroflory pishchevaritel’nogo trakta i organizma (Obligate Symbiosis of Microflora in the Digestive Tract and the Organism), Vilnus: Mokslas, 1989. 6. Skvortsova, E.G., The Role of Proteases of Diet Items and Enteral Microbiota in Digestive Process in Fish of Different Ecological Groups, Extended Abstract of Cand. Sci. (Biol.) Dissertation: Borok, 2002. 7. Ugolev, A.M., Evolyutsiya pishchevareniya i printsipy evolyutsii funktsii (Evolution of the Digestive System and the Principles of Evolution of Functions), Lenin grad: Nauka, 1985. 8. Ugolev, A.M. and Iezuitova, N.N., Determination of the Activity of Invertase and Other Disaccharidases, in Issledovanie pishchevaritel’nogo apparata u cheloveka (obzor sovremennykh metodov) (Study of Human Diges tive Tract (A Review of Modern Methods)), Leningrad: Nauka, 1969, pp. 192–196. INLAND WATER BIOLOGY

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9. Ugolev, A.M. and Kuz’mina, V.V., Pishchevaritel’nye protsessy i adaptatsii u ryb (Digestive Processes and Adaptation in Fish), St. Petersburg: Gidrometeoizdat, 1993. 10. Šyvokiené, J., Simbiontnoe pishchevarenie u gidrobion tov i nasekomykh (Symbiont Digestion in Aquatic Ani mals and Insects), Vilnus: Mokslas, 1989. 11. Anson, M., The Estimation of Pepsin, Tripsin, Papain and Eathepsin with Hemoglobin, J. Gen. Physiol., 1938, vol. 22, no. 1, pp. 79–83. 12. Austin, B., The Bacterial Microflora of Fish, The Sci. World J., 2002, no. 2, pp. 558–572. 13. Bairagi, A., Ghosh, K.S., Sen, S.K., and Ray, A.K., Enzyme Producing Bacterial Flora Isolated from Fish Digestive Tracts, Aquaculture Int., 2002, vol. 10, pp. 109–121. 14. Cahill, M.M., Bacterial Flora of Fishes: A Review, Microbiol. Ecol., 1990, vol. 19, no. 1, pp. 21–41. 15. Clements, K.D., Fermentation and Gastrointestinal Microorganisms in Fishes, in Gastrointestinal Microbio logy, New York, 1997, vol. 1, pp. 156–198. 16. Hansen, G.H. and Olafsen, J.A., Bacterial Interactions in Early Life Stages of Marine Cold Water Fish, Micro biol. Ecol., 1999, vol. 38, pp. 1–26. 17. Hooper, L.V., Bry, L., Falk, P.G., and Gordon, J.I., Host–Microbial Symbiosis in the Mammalian Intes tine: Exploring an Internal Ecosystem, BioEssays, 1998, vol. 20, no. 4, pp. 336–343. 18. Koushik, G., Kumar, S.S., and Kumar, R.A., Charac terization of Bacilli Isolated from the Gut of Rohu, Labeo rohita, Fingerlings and Its Significance in Diges tion, J. Appl. Aquàculture, 2002, vol. 12, no. 3, pp. 33–42. 19. MacDonald, N.L., Stark, J.R., and Austin, B., Bacte rial Microflora in the Gastrointestinal Tract of Dover Sole (Solea solea L.), with Emphasis on the Possible Role of Bacteria in the Nutrition of the Host, FEMS Microbiol. Lett., 1986, vol. 35, pp. 107–111. 20. Ringø, E. and Gatesoupe, F.J., Lactic Acid Bacteria in Fish: A Review, Aquaculture, 1998, vol. 160, pp. 177– 203. 21. Skea, G.L., Mountfort, D.O., and Clements, K.D., Gut Carbohydrases from the New Zealand Marine Herbivo rous Fishes Kyphosus sydneyanus (Kyphosidae), Aplodac tylus arctidens (Aplodactylidae) and Odax pullus (Labridae), Comp. Biochem. Physiol., 2005, vol. 140B, pp. 259–269. 22. Skea, G.L., Mountfort, D.O., and Clements, K.D., Contrasting Digestive Strategies in Four New Zealand Herbivorous Fishes as Reflected by Carbohydrase Activity Profiles, Comp. Biochem. Physiol., 2007, vol. 146A, pp. 63–70. 23. Sugita, H., Kawasaki, J., and Deguchi, Y., Production of Amylase by the Intestinal Microflora in Cultured Freshwater Fish, Lett. Appl. Microbiol., 1997, vol. 24, no. 2, pp. 105–108. 24. Sugita, H., Kawasaki, J., Kumazawa, J., and Deguchi, Y., Production of Amylase by the Intestinal Bacteria of Japanese Coastal Animals, Lett. App. Microbiol., 1996, vol. 23, pp. 174–178. 25. Šyvokiené, J., Michéniené, L., Effect of Heavy Metals on the Bacteriocenoses of the Digestive Tract of Fish, Chemia i inzynieria ekol., 2002, vol. 9, no. 9, pp. 1033– 1038.