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Bacterial flora in the digestive tract of prawns, Penaeus japonicus Bate
Aquaculture, 19 (1980) 229-234 o Elsevier Scientific Publishing Company,
229 Amsterdam
BACTERIAL FLORA IN THE DIGESTIVE PENAE US JAPONICUS BATE
KIMIAKI
YASUDA*
and TADATOSHI
- Printed
in The Netherlands
TRACT OF PRAWNS,
KITAO* *
* Ocean Research Institute, University of Tokyo, l-1 5-1 Minami-dai, Nakano-ku, Tokyo 164 (Japan) ** Department of Agriculture, Miyazaki University, 100 Funatsuka-cho, Miyazaki 880
(Jopan)
(Accepted
16 August
1979)
ABSTRACT Yasuda,
K. and Kitao,
T., 1980. Bacterial
Penaeus japonicus Bate. Aquaculture,
flora in the digestive
tract of prawns,
19: 229-234.
Bacterial flora in the gut of wild cultured prawns, Penaeus japonicus Bate, were investigated and the isolated strains were classified to genera. The flora in the water and mud environment which the prawn inhabits were also investigated. In the water of the prawn_ culture tank, the highest bacterial number was counted in the zoea stages (1.8 x 10’ /ml) and the numbers decreased from the end of the mysis stage to the postlarval stage. Few differences in composition were observed between the two populations. In the digestive tract of the zoea, Vibrio spp. were the dominant genera. In the adult stage, Pseudomonas spp. were the most abundant in the flora of the digestive tract of prawns cultured for 126 days. One prawn that grew poorly had large numbers of the Aeromonas and Vibrio groups in the digestive tract. In the digestive tract of wild prawns, the Pseudomonas group was the dominant bacterial population.
INTRODUCTION
In 1942, Hudinaga developed a hatching and culturing system for the penaeid shrimp, Penaeus juponicus Bate, for the first time. This has been developed into a large-scale prawn cultivation operation in the western part of Japan, and 600,000 kg,of cultured prawns are produced every year. The Japanese Fisheries Administration provides large financial subsidies to produce and stock prawn which are discharged into the sea every year to become one of the major natural fisheries resources of Japan. With the development of prawn cultivation techniques, bacteriological surveys have received more attention because some species of bacteria associated with the prawn cause disease, while other bacteria seem to be a useful food for prawn larvae in large scale cultivation. Several workers have reported the results of quantitative and qualitative investigations on the bacterial population in fish guts (Liston, 1957; Colwell,
230
1962; Aiso et al., 1968; Yoshimizu et al., 1976). However, there have been no studies made on bacteriological surveys in the guts of prawns. In this report we describe the bacterial population in the guts of prawns collected from culture tanks and from Nobeoka Bay, during the period June-October 1973. MATERIALS
AND METHODS
Tanks and rearing water. A small cylindrical tank of 1.5-m diameter and l-m working depth, and a square tank of 16 m2 area and 2-m working depth, were used for hatching and rearing of prawns, respectively. Filtered sea water, which was sterilized using 20 ppm concentration of hypochlorous acid, was placed in the previously cleaned tanks. The sea water in the tanks was dechlorinated with sunlight and aeration before use. Prawns. The prawns used in this experiment were Penaeus japonicus Bate. Mature female prawns caught in Nobeoka Bay were put into the hatching tank in the morning, and during the following night they spawned. The hatched nauplii were transferred to the rearing tank and reared for 126 days. At the zoea stage, Skeletonema costatum was given as food. Rotifers were given as food at the mysis stage. and cuttlefish meal at the adult stage. Samples. Water samples for microbiological analysis were collected from the bay, using a sterile Kitahara sampler, and from the culture tank using sterile glass-stoppered bottles. Mud samples were obtained from the tank with a Phleger-type gravity corer. The body surfaces of prawn larvae were washed first with 20 ppm hypochlorous acid solution for sterilization, then with sterilized sea water. One gram of the larvae was homogenized to isolate attached bacteria. For the isolation of intestinal bacteria, the intestines of 10 cultured and 10 wild prawns were homogenized aseptically before use. The samples were carefully diluted with sterilized artificial sea water prior to inoculating onto plates of medium Trypto-Soi (Eiken, Table I) using sterile spreading rods. Inoculated plates were incubated at 25”C, which
TABLE I Composition
of Trypto-Soi medium
Tryptone “Eiken” Soi-peptone “Eiken” NaCl Agar “Eiken” 70% State sea water Final pH
15g 5fz 5g 15g 1000 ml 9.6
231
was the same temperature as the rearing water, for 2 or 7 days before counting of colonies. kom each plate, 50 colonies were selected and transferred to Trypto-Soi slope medium before the identification of generic level. Identification of isolates. Isolates were pre-incubated on the newly-made Trypto-Soi agar plates and subsequently characterized to genus level using the schemes of Shewan et al. (1960) as modified by Yasuda (1974). The vibriostatic compound O/l 29 (2,4-diamino-6,7-diisopropylpteridine) was not used to distinguish Vibrio from AeFOr?ZO?IUS;the discrimination was made on the basis of the lysin decarboxylase test. RESULTS
The variation in the bacterial numbers in the culture water at the different growth stages of the prawns is shown in F’ig.1. Each count is the mean value of the viable colonies which appeared in the four agar plates made per sample. The highest numbers of bacteria (1.8 X 105/ml) were counted at the zoea stage, and the numbers decreased from the end of the mysis stage to the postlarval stage.
I, , . ,
. , . , . . ,
01
6
(
11
-96 4
Time in days
na@us
zma
my&
post-larvae
adult
M--P
9
mc+w
Stages of growth
Fig.1. Changes in the size of bacterial population measured by viable count (c.f.u./ml) and growth stages of cultured prawn.
232
The generic composition of bacterial populations isolated from the digestive tract of the prawns and from the sea water in the culture tank is shown in Fig.2. Few differences in bacterial composition were observed between these two populations, but in the digestive tract of the zoea stage, which first starts eating bacteria, Vibrio spp. was the dominant genus. During the adult stage, Pseudomonas spp. were the most abundant in the flora of the tract of the prawn cultured for 126 days, while the prawn which grew poorly (about 2 cm body length after 126 days’ cultivation) had many Aeromonas and Vibrio groups in the digestive tract (Fig.3). The digestive tracts of prawns
Seawater
in culture
tank
Digestive
tract
stages of growth
Fig.2. Change of bacterial flora in sea water in the culture tank and in the digestive tract of cultured prawns at different growth stages. Ae, Aeromonas; Vi, Vibrio; I’s, Pseudomonas; Fl, Flavobacterium; St, Staphylococcus; Un, unknown bacteria.
Digestive tract of cultured prawn Body length 2 cm
5 cm
10 cm
Digestive tract of native prawn
Fig.3. Bacterial flora in the digestive tract of cultured prawns after 126 days’ cultivation, and of a wild prawn. The symbols in this figure are as Fig.2, except that both U and Un represent ‘unknown’, and S represents Staphylococcus.
233
which taken from their natural environment had the Pseudomonas group as the dominant bacterial population. After 126 days’ cultivation, the bacterial population of sea water in the tank was dominated by the Vibrio group. Pseudomonas was common at the surface, and in 8 cm depth of sediment, which prawns inhabit at the adult stage. DISCUSSION
The highest numbers of bacteria in the digestive tract of prawns were present when the prawns were in the zoea stage. The numbers then decreased with the age of the prawn from the mysis stage to the postlarval stage (Fig-l). After the eggs of the prawn hatch, and the larvae expend their yolk reserves, they start to feed on Skeletonema sp., followed by bacterial aggregates at the zoea stage. When the balance of prey and predator is maintained in an ecosystem, high population densities are usually observed. In this case, the zoea are consuming the bacterial populations, thus increasing the rate of nutrient regeneration and, therefore, the rate at which they reproduce their cells by using these nutrients (Johannes, 1968). From the mysis stage the prawns start to feed on other micro-organisms larger than bacteria. This may be the reason why bacterial numbers decreased from that stage in the culture tank. Throughout all the larval stages, the generic composition of bacteria in the intestine and in the water of the culture tanks was similar (Fig.2). This implies that the intestinal flora is influenced by external flora throughout the larval stages. After 126 days’ cultivation, the bacterial populations in the tank were dominated by Vibrio spp., but in the sediment, Pseudomonas spp. were dominant. The habitat of the adult prawns is in the sediment. This might imply that the dominant population in the gut was Pseudomonas spp. (Fig.4). Seawater in
culturetank
Bottom seawater in bay
Fig.4. Bacterial flora in the seawater and the sediments in the culture tank, and in bottom seawater in Nobeoka Bay. Mi, Micrococcus. The other symbols in this figure are as Fig. 2.
234
It is interesting to note that healthy cultured adults and wild prawns both had abundant Pseudomonas populations in the gut; if Aeromonas spp. were dominant in the gut, then the prawns showed poor growth. This may imply that Pseudomonas spp. are the most favorable bacterial flora for the healthy growing prawn. However, many Vibrio were isolated from zoea. Although this result is not proved, it is believed that a succession of bacterial populations from zoea to adult stages are important. Further investigations are being carried out to prove this hypothesis. ACKNOWLEDGEMENTS
Thanks are due to Dr. M. Kimura, Department of Agriculture, Miyazaki University, in whose marine station the work was carried out and whose advice and criticism were invaluable. We also wish to thank Dr. N. Taga, Ocean Research Institute, University of Tokyo, for helpful suggestions and critical reading of the manuscript. REFERENCES Aiso, K., Simidu, U. and Hasuo, K., 1968. Microflora in the digestive tract of inshore fish in Japan. J. Gen. Microbial., 52: 361-364. Colwell, R.R., 1962. The bacterial flora of Puget Sound fish. J. Appl. Bacterial., 25: 147158. Hudinaga, H., 1942. Reproduction, development and rearing of Penaeus japonicus Bate. Jpn. J. Zool., 10: 305-393. Johannes, R.E., 1968. Nutrient regeneration in lakes and oceans. Adv. Microbial. Sea., 1: 203-213. Liston, J., 1957. The occurrence and distribution of bacterial types on flatfish. J. Gen. Microbial., 16: 205-216. Shewan, J.M., Hobbs, G. and Hodgkins, W., 1960. The Pseudomonas and Achromobacter groups of bacteria in the spoilage of marine white fish. J. Appl. Bacterial., 23: 463468. Yasuda, K., 1974. Studies on the Role of Marine Bacteria Associated in the Culture Process of Penoeus japonicus. Master’s thesis, University of Miyazaki, 120 pp. Yoshimizu, M., Kimura, T. and Sakai, M., 1976. Studies on the intestinal microflora of salmonids - III. The intestinal microflora of salmon living in the open sea. Bull. Jpn. Sot. Sci. Fish., 42: 875-884:
229 Amsterdam
BACTERIAL FLORA IN THE DIGESTIVE PENAE US JAPONICUS BATE
KIMIAKI
YASUDA*
and TADATOSHI
- Printed
in The Netherlands
TRACT OF PRAWNS,
KITAO* *
* Ocean Research Institute, University of Tokyo, l-1 5-1 Minami-dai, Nakano-ku, Tokyo 164 (Japan) ** Department of Agriculture, Miyazaki University, 100 Funatsuka-cho, Miyazaki 880
(Jopan)
(Accepted
16 August
1979)
ABSTRACT Yasuda,
K. and Kitao,
T., 1980. Bacterial
Penaeus japonicus Bate. Aquaculture,
flora in the digestive
tract of prawns,
19: 229-234.
Bacterial flora in the gut of wild cultured prawns, Penaeus japonicus Bate, were investigated and the isolated strains were classified to genera. The flora in the water and mud environment which the prawn inhabits were also investigated. In the water of the prawn_ culture tank, the highest bacterial number was counted in the zoea stages (1.8 x 10’ /ml) and the numbers decreased from the end of the mysis stage to the postlarval stage. Few differences in composition were observed between the two populations. In the digestive tract of the zoea, Vibrio spp. were the dominant genera. In the adult stage, Pseudomonas spp. were the most abundant in the flora of the digestive tract of prawns cultured for 126 days. One prawn that grew poorly had large numbers of the Aeromonas and Vibrio groups in the digestive tract. In the digestive tract of wild prawns, the Pseudomonas group was the dominant bacterial population.
INTRODUCTION
In 1942, Hudinaga developed a hatching and culturing system for the penaeid shrimp, Penaeus juponicus Bate, for the first time. This has been developed into a large-scale prawn cultivation operation in the western part of Japan, and 600,000 kg,of cultured prawns are produced every year. The Japanese Fisheries Administration provides large financial subsidies to produce and stock prawn which are discharged into the sea every year to become one of the major natural fisheries resources of Japan. With the development of prawn cultivation techniques, bacteriological surveys have received more attention because some species of bacteria associated with the prawn cause disease, while other bacteria seem to be a useful food for prawn larvae in large scale cultivation. Several workers have reported the results of quantitative and qualitative investigations on the bacterial population in fish guts (Liston, 1957; Colwell,
230
1962; Aiso et al., 1968; Yoshimizu et al., 1976). However, there have been no studies made on bacteriological surveys in the guts of prawns. In this report we describe the bacterial population in the guts of prawns collected from culture tanks and from Nobeoka Bay, during the period June-October 1973. MATERIALS
AND METHODS
Tanks and rearing water. A small cylindrical tank of 1.5-m diameter and l-m working depth, and a square tank of 16 m2 area and 2-m working depth, were used for hatching and rearing of prawns, respectively. Filtered sea water, which was sterilized using 20 ppm concentration of hypochlorous acid, was placed in the previously cleaned tanks. The sea water in the tanks was dechlorinated with sunlight and aeration before use. Prawns. The prawns used in this experiment were Penaeus japonicus Bate. Mature female prawns caught in Nobeoka Bay were put into the hatching tank in the morning, and during the following night they spawned. The hatched nauplii were transferred to the rearing tank and reared for 126 days. At the zoea stage, Skeletonema costatum was given as food. Rotifers were given as food at the mysis stage. and cuttlefish meal at the adult stage. Samples. Water samples for microbiological analysis were collected from the bay, using a sterile Kitahara sampler, and from the culture tank using sterile glass-stoppered bottles. Mud samples were obtained from the tank with a Phleger-type gravity corer. The body surfaces of prawn larvae were washed first with 20 ppm hypochlorous acid solution for sterilization, then with sterilized sea water. One gram of the larvae was homogenized to isolate attached bacteria. For the isolation of intestinal bacteria, the intestines of 10 cultured and 10 wild prawns were homogenized aseptically before use. The samples were carefully diluted with sterilized artificial sea water prior to inoculating onto plates of medium Trypto-Soi (Eiken, Table I) using sterile spreading rods. Inoculated plates were incubated at 25”C, which
TABLE I Composition
of Trypto-Soi medium
Tryptone “Eiken” Soi-peptone “Eiken” NaCl Agar “Eiken” 70% State sea water Final pH
15g 5fz 5g 15g 1000 ml 9.6
231
was the same temperature as the rearing water, for 2 or 7 days before counting of colonies. kom each plate, 50 colonies were selected and transferred to Trypto-Soi slope medium before the identification of generic level. Identification of isolates. Isolates were pre-incubated on the newly-made Trypto-Soi agar plates and subsequently characterized to genus level using the schemes of Shewan et al. (1960) as modified by Yasuda (1974). The vibriostatic compound O/l 29 (2,4-diamino-6,7-diisopropylpteridine) was not used to distinguish Vibrio from AeFOr?ZO?IUS;the discrimination was made on the basis of the lysin decarboxylase test. RESULTS
The variation in the bacterial numbers in the culture water at the different growth stages of the prawns is shown in F’ig.1. Each count is the mean value of the viable colonies which appeared in the four agar plates made per sample. The highest numbers of bacteria (1.8 X 105/ml) were counted at the zoea stage, and the numbers decreased from the end of the mysis stage to the postlarval stage.
I, , . ,
. , . , . . ,
01
6
(
11
-96 4
Time in days
na@us
zma
my&
post-larvae
adult
M--P
9
mc+w
Stages of growth
Fig.1. Changes in the size of bacterial population measured by viable count (c.f.u./ml) and growth stages of cultured prawn.
232
The generic composition of bacterial populations isolated from the digestive tract of the prawns and from the sea water in the culture tank is shown in Fig.2. Few differences in bacterial composition were observed between these two populations, but in the digestive tract of the zoea stage, which first starts eating bacteria, Vibrio spp. was the dominant genus. During the adult stage, Pseudomonas spp. were the most abundant in the flora of the tract of the prawn cultured for 126 days, while the prawn which grew poorly (about 2 cm body length after 126 days’ cultivation) had many Aeromonas and Vibrio groups in the digestive tract (Fig.3). The digestive tracts of prawns
Seawater
in culture
tank
Digestive
tract
stages of growth
Fig.2. Change of bacterial flora in sea water in the culture tank and in the digestive tract of cultured prawns at different growth stages. Ae, Aeromonas; Vi, Vibrio; I’s, Pseudomonas; Fl, Flavobacterium; St, Staphylococcus; Un, unknown bacteria.
Digestive tract of cultured prawn Body length 2 cm
5 cm
10 cm
Digestive tract of native prawn
Fig.3. Bacterial flora in the digestive tract of cultured prawns after 126 days’ cultivation, and of a wild prawn. The symbols in this figure are as Fig.2, except that both U and Un represent ‘unknown’, and S represents Staphylococcus.
233
which taken from their natural environment had the Pseudomonas group as the dominant bacterial population. After 126 days’ cultivation, the bacterial population of sea water in the tank was dominated by the Vibrio group. Pseudomonas was common at the surface, and in 8 cm depth of sediment, which prawns inhabit at the adult stage. DISCUSSION
The highest numbers of bacteria in the digestive tract of prawns were present when the prawns were in the zoea stage. The numbers then decreased with the age of the prawn from the mysis stage to the postlarval stage (Fig-l). After the eggs of the prawn hatch, and the larvae expend their yolk reserves, they start to feed on Skeletonema sp., followed by bacterial aggregates at the zoea stage. When the balance of prey and predator is maintained in an ecosystem, high population densities are usually observed. In this case, the zoea are consuming the bacterial populations, thus increasing the rate of nutrient regeneration and, therefore, the rate at which they reproduce their cells by using these nutrients (Johannes, 1968). From the mysis stage the prawns start to feed on other micro-organisms larger than bacteria. This may be the reason why bacterial numbers decreased from that stage in the culture tank. Throughout all the larval stages, the generic composition of bacteria in the intestine and in the water of the culture tanks was similar (Fig.2). This implies that the intestinal flora is influenced by external flora throughout the larval stages. After 126 days’ cultivation, the bacterial populations in the tank were dominated by Vibrio spp., but in the sediment, Pseudomonas spp. were dominant. The habitat of the adult prawns is in the sediment. This might imply that the dominant population in the gut was Pseudomonas spp. (Fig.4). Seawater in
culturetank
Bottom seawater in bay
Fig.4. Bacterial flora in the seawater and the sediments in the culture tank, and in bottom seawater in Nobeoka Bay. Mi, Micrococcus. The other symbols in this figure are as Fig. 2.
234
It is interesting to note that healthy cultured adults and wild prawns both had abundant Pseudomonas populations in the gut; if Aeromonas spp. were dominant in the gut, then the prawns showed poor growth. This may imply that Pseudomonas spp. are the most favorable bacterial flora for the healthy growing prawn. However, many Vibrio were isolated from zoea. Although this result is not proved, it is believed that a succession of bacterial populations from zoea to adult stages are important. Further investigations are being carried out to prove this hypothesis. ACKNOWLEDGEMENTS
Thanks are due to Dr. M. Kimura, Department of Agriculture, Miyazaki University, in whose marine station the work was carried out and whose advice and criticism were invaluable. We also wish to thank Dr. N. Taga, Ocean Research Institute, University of Tokyo, for helpful suggestions and critical reading of the manuscript. REFERENCES Aiso, K., Simidu, U. and Hasuo, K., 1968. Microflora in the digestive tract of inshore fish in Japan. J. Gen. Microbial., 52: 361-364. Colwell, R.R., 1962. The bacterial flora of Puget Sound fish. J. Appl. Bacterial., 25: 147158. Hudinaga, H., 1942. Reproduction, development and rearing of Penaeus japonicus Bate. Jpn. J. Zool., 10: 305-393. Johannes, R.E., 1968. Nutrient regeneration in lakes and oceans. Adv. Microbial. Sea., 1: 203-213. Liston, J., 1957. The occurrence and distribution of bacterial types on flatfish. J. Gen. Microbial., 16: 205-216. Shewan, J.M., Hobbs, G. and Hodgkins, W., 1960. The Pseudomonas and Achromobacter groups of bacteria in the spoilage of marine white fish. J. Appl. Bacterial., 23: 463468. Yasuda, K., 1974. Studies on the Role of Marine Bacteria Associated in the Culture Process of Penoeus japonicus. Master’s thesis, University of Miyazaki, 120 pp. Yoshimizu, M., Kimura, T. and Sakai, M., 1976. Studies on the intestinal microflora of salmonids - III. The intestinal microflora of salmon living in the open sea. Bull. Jpn. Sot. Sci. Fish., 42: 875-884: