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Luminous microflora associated with the fishes Mugil cephalus and Tachysurus arius
FEMS Microbiology Ecology 53 (1988) 27-34 Published by Elsevier
27
FEC 00145
Luminous microflora associated with the fishes Mugil cephalus and Tachysurus arius A. Ramesh
a
and V.K. Venugopalan b
National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta-700010, and b Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai - 608 502, Tamil Nadu, India
Key words: Luminous bacterium; Vibrio sp.; Mugil cephalus; Tachysurus arius
1. S U M M A R Y Luminous bacteria harboured in the skin, gill and gut of the fishes Mugil cephalus and Tachysurus arius were studied. Within the gut, the distribution of bacteria was studied regionwise, i.e., foregut, midgut and hindgut. In M. cephalus, m a x i m u m luminous bacterial population density was observed in the hindgut and minimum was found in the foregut. In T. arius, m a x i m u m luminous bacterial population density was recorded in the hindgut and minimum was found in the midgut. Luminous microflora associated with the host showed seasonal variation. Bacterial load of the surrounding medium and type of food governed the distribution of luminous microbiota in fish. Vibrio haroeyi and V. fischeri were the two species identified, the former accounting for the majority of the isolates.
2. I N T R O D U C T I O N G r o w t h and distribution of heterotrophic bacteria are influenced by the availability of nutrients. Hence, the heterotrophic luminous bacteria
Correspondence to: A. Ramesh, Microbiology Division, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme XM, Beliaghata, Calcutta - 700010, India.
are found in association with estuarine and marine organisms, as these hosts provide them with an increased concentration of nutrients, like macromolecules and other organic materials essential for growth [1]. Therefore, the present study was aimed to provide information about the distribution profiles of luminous bacteria harboured on the external surface as well as within the body of the host fish.
3. M A T E R I A L S A N D M E T H O D S The fishes Mugil cephalus and Tachysurus arius were sampled from the Vellar estuary for a period of one year (from April 1981 to March 1982). They were brought alive to the laboratory in polythene buckets immediately after collection. For the isolation of bacteria from the skin, the method of Potter and Baker [2] was followed. Sterilized cotton swabs were used to collect the bacteria from the skin and the gills of the abovementioned fishes and were dipped in sterile seawater to transfer the inoculum. Appropriate dilutions of the inoculum were made for skin and gill samples and then plated in duplicate onto seawater complex agar (SWC) [3] medium. For screening the bacteria of the gut, the fish surface was washed several times with sterile seawater to prevent contamination from the skin.
0168-6496/88/$03.50 © 1988 Federation of European Microbiological Societies
28 T h e gut of the fish was then removed aseptically a n d divided into foregut ( o e s o p h a g u s a n d stomach), midgut (anterior half of the intestine) and hindgut (posterior half of the intestine). The three gut sections were then opened and shaken in 100 ml sterile seawater with the help of a mechanical gyratory shaker, allowed to settle and the supernatant was carefully removed. Serial dilutions were m a d e using 1 ml of the liquid and 9 ml of sterile seawater and plated in duplicate o n t o S W C agar medium. The spread plate m e t h o d was followed to spread the inoculum. Details regarding inoculation of plates, counting of colony forruing units (CFU), maintenance and identification of the isolates have already been described elsewhere [4]. Population densities were calculated on the basis of dry weight of gut content, wet weight of gill and cm 2 area of skin in the case of gut, gill and skin, respectively.
Table 1 Percentage of luminous CFU among total CFU associated with Mugil cephalus Month
Region
April 1981 May June July August September October November December January 1982 February March
Skin
Gill
Gut
46.2 61.7 24.2 64.1 44.1 67.4 0 0 23.3 53.0 37.6 53.3
59.3 67.5 22.3 82.2 46.5 88.4 0 0 21.8 74.2 33.5 71.2
79.3 23.0 64.2 35.6 13.2 68.8 0 0 58.7 70.5 74.6 57.1
Gut
lO0
4. R E S U L T S
80
6C
The results are discussed on a seasonal basis. The entire sampling period was divided into four seasons and the values for the three m o n t h s of a season are pooled. The seasons are summer (April to June), p r e m o n s o o n (July to September), m o n s o o n (October to December) and postm o n s o o n (January to March). 4.1. Mugil cephalus 4.1.1. Skin and gill. F o r b o t h skin and gill the m i n i m u m count of luminous bacteria was rec o r d e d during the m o n s o o n period and the maxim u m during the p r e m o n s o o n season. The luminous microfloral count fluctuated from 1.3 • 104 to 3.6. 104 C F U / c m 2 in skin and 9 . 1 . 1 0 3 to 5 . 1 - 1 0 4 C F U / g wet wt. in gill. The luminous microbiota constituted 23.3-67.4% of the total heterotrophic bacterial population in skin, 21.8-88.4% of the same in gill (Table 1). L u m i n o u s microbes were n o t detected from the skin and gill in the m o n t h s of O c t o b e r and November. Species composition of the isolates revealed the occurrence of V. harveyi (78% in the skin, 92% in the gill) and V. fischeri (22% in the skin and 8% in the gill) (Fig. 1). 4.1.2. Gut. As observed in the case of the skin
40 2C 0 Gill
100
~ 8D "5 60
~o ~. 2o 0
lOC 80
60 z.O 20 0 A
M
J 1981
3
A
S MONTHS
0
N
O
J
F
M
]a82
Fig. 1. Species composition of luminous bacteria associated with skin, gill and gut of M. cephalus. I, V. harveyi; [], V. fischeri. Months are indicated by their first letters.
29
and the gill, the luminous bacterial load was also low in the gut during the monsoon season but a high bacterial load was noted during the postmonsoon season (Table 1). Within the gut, the foregut and midgut, but not the hindgut, showed minimum luminous counts during the monsoon season (Table 2). All the three gut regions showed maximum luminous bacterial population during the postmonsoon season. In the gut, the values for the luminous bacterial population ranged from 4.3-105 to 6 . 3 - 1 0 3 C F U / g dry weight of contents. Within the gut, the luminous CFU ranged from 2.1.104 to 5.1105 C F U / g dry wt. of contents in the foregut. In the midgut, the CFU ranged from 2.0.10 4 tO 3 . 5 - 1 0 5 / g dry wt. of contents, whereas in the hindgut the values fluctuated from 1.9-10 4 to 4.4" 105 C F U / g dry wt. of contents. As observed in the case of skin and gills the luminous microbiota was not detected in the gut region in October and November. The luminous CFU in the gut ranged from 13.2 to 79.3% of the total CFU (Table 1). In the three gut regions, the luminous microbial counts fluctuated from 26.0 to 74.6% in the foregut; from 17.7 to 87.0% in the midgut and 22.8 to 99.6% in the hindgut (Table 2). On an annual basis V. harveyi and V. fischeri accounted for 78.5 and 21.5% of the isolates, respectively, in the gut. Unlike V. fischeri, V.
Table 2 Percentage of luminous C F U a m o n g total C F U within the gut of Mugil cephalus Month
Foregut
Midgut
Hindgut
April 1981 May June July August September October November December January 1982 February March
57.7 27.3 61.2 26.0 0 47.6 0 0 1.6 69.1 74.6 59.2
18.7 17.7 87.0 31.1 0 53.8 0 0 47.8 74.6 67.3 67.5
53.0 22.8 57.6 62.7 40.2 89.3 0 0 99.6 69.0 82.0 47.0
harveyi was recorded continuously in all the regions of gut (Fig. 1). The quantitative distribution of luminous microflora was studied in relation to the type of food. A maximum luminous bacterial load was encountered during the postmonsoon season when algal filaments and diatoms dominated the diet. However, the same components with diatoms in lesser proportions were found in the diet of mullet (M. cephalus) during the monsoon season when the luminous bacterial load was also minimum. A negative relationship was observed between dry weight of the gut contents and luminous bacterial population in the gut (Fig. 2). 4.2. Tachysurus arius 4.2.1. Skin and gill. Minimum luminous bacterial count was recorded during the monsoon season, whereas the maximum was during the summer season. The luminous microfloral count ranged from 1.5.10 4 to 4.8.103 C F U / / c m 2 i n skin and 3.4.10 4 to 6.2" 1 0 3 C F U / g wet wt. in the gill. Skin and gill were devoid of luminous bacteria in October and November. The percentage composition of luminous microfloral population ranged from 8.4 to 84.3% of the total C F U on skin and 10.7 to 99.9% of the same in the gill (Table 3). On an annual basis V. harveyi constituted 84% of the luminous isolates on the skin and 82% in the gill, whereas V. fischeri accounted for 16% of the isolates in the skin and 18% in the gill (Fig. 3). 4.2.2. Gut. The luminous microfloral counts were less in the gut (in all the regions) during the monsoon season. But a high bacterial load was recorded during the postmonsoon season as in the case of skin and the gill (Tables 3 and 4). In the gut, the luminous microfloral counts varied from 2 .6 • 105 to 3.6.104 C F U / g dry wt. of contents of the three gut regions, the luminous bacterial count in the foregut ranging from 6.9.102 to 1.6-104 C F U / g dry wt. In the midgut, the count ranged from 3.4.104 to 3.7- 105 C F U / g dry wt. of gut contents, whereas in the hindgut the value varied from 3.2.104 to 5.3- 105 C F U / g dry wt. The luminous bacterial count in different regions of the gut ranged from 14.9 to 87.1% of the total bacterial count in the foregut, 8.6 to 88.6% in
30 6
Table 3
a
""-
5
/\z
\
Percentage of luminous CFU among total CFU associated with Tachysurus arius
.
Month
4
Region Skin
Gill
Gut
41.7 64.2 55.6 39.5 39.3 84.3 0 0 8.4 41.0 73.7 81.4
86.8 41.3 10.7 34.7 14.5 93.0 0 0 52.9 27.3 67.5 99.9
69.2 58.6 25.1 42.3 55.3 71.5 0 0 49.6 70.4 84.4 75.8
A
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i
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i
t,._ II)
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S 0
'
l
i
N D J
J
F M
b z3
O t-
6
5
•\ / \ \
?=
o u
s "~ Z ._~
April 1981 May June July August September October November December January 1982 February March
./\ \.~"
•
t-"XD
""-"
.4
Gut
O
GI O
100
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i i A,
"'~
t
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A
i
i
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i
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i
i
i
N D J
i
F M
1982
1981 MONTHS
Fig. 2. Relationship between luminous bacterial population and dry weight of gut contents in M. cephalus (a) and T. arius (b). Here, gut bacterial populations were given as log CFU which is actually log of C F U / g dry weight of gut contents versus log of total dry weight of gut contents plotted against the sampling period. Months are indicated by their first letters.
8[
o: Skin ~13C
the midgut and 21.2 to 97.0% in the hindgut (Table 4). On an annual basis V. harveyi accounted for 76.8% of the isolates and II. fischeri 23.2% (Fig. 3). When gut content analysis of the fish was compared with the luminous bacterial load, the following trend was evident. Maximum luminous bacterial population was recorded in the postmonsoon season in T. arius when crustaceans dominated the diets. Minimum values were encountered during the monsoon season when fish,
8C
6{
Z.( 2
A
M
1981
J
J
A
S MONTHS
0
N
D
J
F
M
1982 Fi 9
"{
Fig. 3. Qualitative variation of luminous bacteria associated with skin, gill and gut of T. arius. Filled bars, V. haroeyi; hatched bars, V. fischeri. Months are indicated by their first letters.
31 Table 4 Percentage of luminous CFU among total CFU within the gut of Tachysurus arius Month
Foregut
Midgut
Hindgut
April 1981 May June July August September October November December January 1982 February March
65.1 58.0 77.9 76.7 14.9 57.4 0 0 39.5 69.6 82.6 87.1
64.8 37.7 8.6 8.6 88.1 82.4 0 0 45.5 88.6 71.8 64.3
82.3 21.2 90.4 76.7 74.4 83.5 0 0 63.4 72.3 97.0 78.3
polychaetes etc. formed the main food constituents. A negative relationship was observed between the weight of the food and luminous bacterial load in the gut (Fig. 2).
5. D I S C U S S I O N The three sites, namely the skin, the gill and the gut were selected in the two species of fish for studying the distribution and seasonal variation of the luminous microfloral population, because they offer ideal microenvironments [5] for the growth of these bacteria. The fishes M. cephalus and T. arius were chosen on the basis of their feeding habits. The former is a detritivore whereas the latter is a carnivore. They were selected to ascertain whether there is any relationship between the distribution of luminous procaryotes and the type of food consumed by these fishes. The extent of distribution of luminous bacteria a m o n g the total bacterial population (CFU) is quite interesting, 18-54% of the bacterial colonies isolated from the gut of Argyropelecus hemigymnus, Oxyjulis californica and Chromis punctipinnis being photogenic [6]. O'Brien and Sizemore [7] found that 0.44 and 20.55% of the total viable counts from stomach and intestine, respectively, were luminous. On the whole, the findings of the present investigation revealed that the luminous
microbiota (harboured in skin, gill and gut) constitute a significant portion of the total heterotrophic bacterial population with the luminous counts at times reaching up to 84% of the total counts. Maximum luminous counts were recorded in the skin of M. cephalus and T. arius during premonsoon and summer seasons, respectively, and minimum counts during the monsoon season. Generally, the skin of fishes is easily accessible to the bacterial flora of the surrounding water, and after colonization the bacteria thrive well on the slime secreted by the fish. The luminous bacterial density in the Vellar estuary was also correspondingly low (Table 5) during the monsoon period and hence a change in salinity of the water was reflected in the luminous bacterial count in the skin of the fish as well. The luminous microfloral population in the skin of M. cephalus was proportionately greater than that in T. arius. Since M. cephalus is a bottom feeder, the chances of this fish getting contaminated with sediment bacteria [8] cannot be ruled out and this may account for the higher incidence of microflora found on the skin. Horsley [9] was also of the opinion that fish inhabiting or in contact with the mud would get contaminated
Table 5 Water salinity and luminous bacterial population of water and sediment of the Vellar estuary Month
April 1981 May June July August September October November December January 1982 February March
Water salinity (%0)
Luminous CFU Water (CFU/ml)
Sediment ( C F U / g / d r y wt.)
32.3 30.6 31.8 21.6 30.5 32.0 17.0 18.0 21.7
31.5 33.1 18.5 13.5 6.7 19.9 6.4 3.5 3.5
32.4.103 27.3.103 24.3.103 16.9.103 17.4.103 10.2.103 51.0.102 50.6.10 2 85.1.102
29.4 28.9 29.2
25.1 24.1 31.3
16.8.103 12.5.103 23.1.103
32 from sediment microflora. In the present study, the maximum luminous bacterial load in the gill of M. cephalus was detected during the premonsoon season and in the gill of T. arius during the summer season. As observed in the case of skin, the minimum luminous bacterial density was recorded in the monsoon season in the gills of both fish. This can be ascribed to the depletion in luminous bacterial population in the surrounding medium during that period (Table 5). Since the gills are constantly exposed to the movement of water, any change in the bacterial density of the water should be reflected in the gill microfloral population also. Hence, it would be reasonable to assume that the low luminous bacterial population observed in the gill is indicative of the changes in the population size of the luminous bacteria in the water. Although salinity likely influences the size of the luminous bacterial population of the skin and the gill as a whole, minor variations in the numerical abundance of the luminous bacteria encountered could also be due to factors such as secretion of antimicrobial substances from the skin [10], tissue integrity, secretory bactericidins and production of mucus [11]. Data collected in the present study indicated that the luminous bacterial density of the gut in both the fishes (like that of the skin and gill) was influenced by seasonal changes. Since salinity seems to influence the luminous bacterial density in water (Table 5) it may indirectly influence the bacterial load of the gut also, because the fish secures its food from the water and sediment. Since M. cephalus and T. arius secure their food from the sediment and water, respectively, both of which recorded a very low luminous bacterial population in the monsoon months due to the effect of low salinity, it would be reasonable to infer that salinity indirectly influenced the bacterial load of the gut. Salinity-induced seasonal variations, similar to those in the present study, in the enteric luminous microflora of milk fish (Chanos chanos) have already been reported by Ramesh et al. [4]. The variations in the number of luminous bacteria in gut could be attributed to (a) the rate of emptying of the stomach which depends on food type, meal size, fish size and frequency of
feeding. Frequency of feeding in turn depends on texture, palpability of the food, digestibility and its energy and nutrient content [12]; (b) retention or residence time of food material in the gut [13] and (c) other factors such as previous feeding history of the animal [6] and its fatness and reproductive status [12]. These may also have a bearing on the gut microflora of the fish. Maximum luminous bacterial population was recorded in the alimentary tract of M. cephalus, in the postmonsoon season, when algal filaments and diatoms were the main constituents of the diet. Algal filaments are known to secrete metabolites which nourish the epiphytic microflora [14]. Lakshmanaperumalsamy and Purushothaman [15] reported the association of heterotrophic bacteria with seaweeds of the Vellar estuary. It is possible that, in M. cephalus, low-molecular-weight metabolites released from plankton and algae during the course of digestion could foster these bacterial populations. Though the same food components were encountered in the diet of mullet during the monsoon season also (when minimum luminous bacterial population was found), sand grains were greater in quantity. Maximum luminous bacterial load was registered in the gut of T. arius when crustaceans formed the major constituents of the diet. The association of luminous bacteria with crustaceans was documented by Baross et al. [16] and Sochard et al. [17]. Proliferation of luminous microbes might have taken place in the gut of T. arius as a result of the large amount of chitin available during that season. Hastings and Nealson [18] reported production of chitinase by luminous bacteria. Minimum counts were recorded when crustaceans were present in lesser proportions in the diet. Our observations concurred with those of O'Brien and Sizemore [7] who proved experimentally that fish fed with chitin-supplemented food registered greater counts of luminous bacteria than fish fed with food lacking chitin. Hence, the crustacean diet may account for the relative abundance of luminous bacteria in the gut of T. arius compared to that in M. cephalus. Attempts were also made to relate the luminous bacterial density in the gut of M. cephalus and T. arius to the weight of food present in the gut (Fig.
33 2). The negative relationship encountered suggested that the size of luminous microflora did not d e p e n d on the weight of the food alone. The findings of the present study are in contrast to those of R u b y and Morin [6] and O'Brien and Sizemore [7] who reported a relationship between the bacterial population and the feeding intensity of fish. It is also possible that the population size of the luminous microflora depends on the relative weight of various items in the diet rather than on the absolute weight of the food. The population density of luminous microbiota was greater in the gut than in the other microenvironments, viz., skin and gill, thus supporting the hypothesis of Horsley [9] that the fish gut acts as an enrichment vessel for the growth of bacteria. I n view of the extensive colonization of the digestive tract by the luminous procaryotes, the question naturally arises as to the possible function of these bacteria in the gut. The prime role of luminous bacteria in the gut of fish would appear to be nutritional, since all luminous procaryotes elaborate chitinase which degrades chitin in the gut into N-acetylglucosamine [19]. Peres et al. [20] f o u n d absorption o f N-acetylglucosamine in fish gut. V. haroeyi w a s the d o m i n a n t species in the gut, skin and gill of b o t h fishes. This was followed by V. fischeri. Preponderance of V. haroeyi in the gut was reported by Nair et al. [21], O'Brien and Sizemore [7] and R a m e s h et al. [22]. The main rationale for the distribution of V. harveyi might be its nutritional versatility [19] as it is able to utilize between 30 and 45 organic c o m p o u n d s (depending u p o n the strain). V. fischeri can utilise 7 - 2 2 and P. leiognathi only 15 c o m p o u n d s as nutrients. A n o t h e r plausible reason that could be advanced to explain the association of V. harveyi with fish is its flagellation. V. harveyi possesses lateral flagella, whereas polar flagellation is exhibited by V. fischeri and P. leiognathi. Belas and Colwell [23] studied the adsorption of laterally and polarly flagellated vibrios to chitin using the L a n g m u i r adsorption isotherm. T h e y reported a direct correlation between the lateral flagella production, surface binding nature of the flagella and the selective inhibition of polarly flagellated
bacteria by laterally flagellated bacteria. A l t h o u g h it was d e m o n s t r a t e d for V. parahaemolyticus, a species closely related to V. haroeyi, the same was likely to hold g o o d in the case of V. haroeyi, since its lateral flagella provide enough surface area for adsorption. McCall and Sizemore [24] screened a bacteriocin called ' h a r v e y c i n ' synthesized by V. harveyi which was k n o w n to be lethal to closely related bacteria. ~Bacteriocinogeny bestows a competitive advantage on V. harveyi to d o m i n a t e over nonbacteriocinogenic strains.
REFERENCES [1] Costerton, J.W. and Geesey, G.G. (1979) Microbial contamination of surfaces. In: Surface Contamination (Mittal, K.L., Ed.), Vol. I, pp. 211-221. Plenum Publishing Corporation, New York. [2] Potter, L.F. and Baker, G.E. (1961) The role of fish as conveyors of microorganisms in aquatic environments. Can. J. Microbiol. 7, 595-605. [3] Ruby, E.G. and Nealson, K.H. (1978) Seasonal changes in species composition of luminous bacteria in near shore seawater. Limnol. Oceanogr. 23, 530-533. [4] Ramesh, A., Nandakumar, R. and Venugopalan, V.K. (1986) Enteric luminous microflora of the pond-cultured mill
34
[13]
[14]
[15]
[16]
[17]
Control Processes in Fish Physiology (Ranklin, J.C., Pitcher, T.J. and Duggan, R.T., Eds.), pp. 23-40, Croom Helm Ltd., London. Hood, M.A. and Meyers, S.P. (1973) Microbial aspects of Penaeid shrimp digestion. Proc. Gulf and Caribbean fish. Inst., 26th Annual Session, October 1973, pp. 81-92. Fogg, G.E. (1966) Extracellular products of algae. In: Oceanogr. Mar. Biol. Annu. Rev. (Barnes, H., Ed.), pp. 195-212. George Allen and Unwin Ltd., London. Lakshmanaperumalsamy, P. and Purushothaman, A. (1982) Heterotrophic bacteria associated with seaweed. Proc. Indian Acad. Sci. (Plant Sei). 91,487-493. Baross, J.A., Tester, P.A. and Morita, R.Y. (1978) Incidence, microscopy and etiology of exoskeleton lesions in the tanner crab, Chinoecetes tanneri. J. Fish. Res. Bd. Can. 35, 1141-1149. Sochard, M.R., Wilson, D.F., Austin, B. and Colwell, R.R. (1979) Bacteria associated with the surface and gut of marine copepods. Appl. Environ. Microbiol. 37, 750-759.
[18] Hastings, J.W. and Nealson, K.H. (1977) Bacterial bioluminescence. Annu. Rev. Microbiol. 31, 549-595. [19] Reichelt, J.L. and Baumann, P. (1973) Taxonomy of the marine luminous bacteria. Arch. Microbioi. 94, 283-330. [20] Peres, G., Boge, B., Colin, D. and Rigal, A. (1973) Effects of temperature on the fish digestive process. Enzymic activities and intestinal absorption. Rev. Trab. Inst. (Sci. Yech.) Pech. Marit. 37, 223-232. [21] Nair, G.B., Abraham, M. and Natarajan, R. (1979) Isolation and identification of luminous bacteria from Porto Novo estuarine environs. Indian J. Mar. Sci. 8, 46-48. [22] Ramesh, A., Loganathan, B. and Venugopalan, V.K. (1985) Enteric luminous bacteria of bivalves from Porto Novo waters. J. Singapore Nat. Acad. Sci. 14, 116-119. [23] Belas, M.R. and Colwell, R.R. (1982) Adsorption kinetics of laterally and polarly flagellated Vibrio. J. Bacteriol. 151, 1568-1580. [24] McCall, J.O. and Sizemore, R.K. (1979) Description of a bacteriocinogenic plasmid in Beneckea harveyi Appl. Environ. Microbiol. 38, 974-979.
27
FEC 00145
Luminous microflora associated with the fishes Mugil cephalus and Tachysurus arius A. Ramesh
a
and V.K. Venugopalan b
National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta-700010, and b Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai - 608 502, Tamil Nadu, India
Key words: Luminous bacterium; Vibrio sp.; Mugil cephalus; Tachysurus arius
1. S U M M A R Y Luminous bacteria harboured in the skin, gill and gut of the fishes Mugil cephalus and Tachysurus arius were studied. Within the gut, the distribution of bacteria was studied regionwise, i.e., foregut, midgut and hindgut. In M. cephalus, m a x i m u m luminous bacterial population density was observed in the hindgut and minimum was found in the foregut. In T. arius, m a x i m u m luminous bacterial population density was recorded in the hindgut and minimum was found in the midgut. Luminous microflora associated with the host showed seasonal variation. Bacterial load of the surrounding medium and type of food governed the distribution of luminous microbiota in fish. Vibrio haroeyi and V. fischeri were the two species identified, the former accounting for the majority of the isolates.
2. I N T R O D U C T I O N G r o w t h and distribution of heterotrophic bacteria are influenced by the availability of nutrients. Hence, the heterotrophic luminous bacteria
Correspondence to: A. Ramesh, Microbiology Division, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme XM, Beliaghata, Calcutta - 700010, India.
are found in association with estuarine and marine organisms, as these hosts provide them with an increased concentration of nutrients, like macromolecules and other organic materials essential for growth [1]. Therefore, the present study was aimed to provide information about the distribution profiles of luminous bacteria harboured on the external surface as well as within the body of the host fish.
3. M A T E R I A L S A N D M E T H O D S The fishes Mugil cephalus and Tachysurus arius were sampled from the Vellar estuary for a period of one year (from April 1981 to March 1982). They were brought alive to the laboratory in polythene buckets immediately after collection. For the isolation of bacteria from the skin, the method of Potter and Baker [2] was followed. Sterilized cotton swabs were used to collect the bacteria from the skin and the gills of the abovementioned fishes and were dipped in sterile seawater to transfer the inoculum. Appropriate dilutions of the inoculum were made for skin and gill samples and then plated in duplicate onto seawater complex agar (SWC) [3] medium. For screening the bacteria of the gut, the fish surface was washed several times with sterile seawater to prevent contamination from the skin.
0168-6496/88/$03.50 © 1988 Federation of European Microbiological Societies
28 T h e gut of the fish was then removed aseptically a n d divided into foregut ( o e s o p h a g u s a n d stomach), midgut (anterior half of the intestine) and hindgut (posterior half of the intestine). The three gut sections were then opened and shaken in 100 ml sterile seawater with the help of a mechanical gyratory shaker, allowed to settle and the supernatant was carefully removed. Serial dilutions were m a d e using 1 ml of the liquid and 9 ml of sterile seawater and plated in duplicate o n t o S W C agar medium. The spread plate m e t h o d was followed to spread the inoculum. Details regarding inoculation of plates, counting of colony forruing units (CFU), maintenance and identification of the isolates have already been described elsewhere [4]. Population densities were calculated on the basis of dry weight of gut content, wet weight of gill and cm 2 area of skin in the case of gut, gill and skin, respectively.
Table 1 Percentage of luminous CFU among total CFU associated with Mugil cephalus Month
Region
April 1981 May June July August September October November December January 1982 February March
Skin
Gill
Gut
46.2 61.7 24.2 64.1 44.1 67.4 0 0 23.3 53.0 37.6 53.3
59.3 67.5 22.3 82.2 46.5 88.4 0 0 21.8 74.2 33.5 71.2
79.3 23.0 64.2 35.6 13.2 68.8 0 0 58.7 70.5 74.6 57.1
Gut
lO0
4. R E S U L T S
80
6C
The results are discussed on a seasonal basis. The entire sampling period was divided into four seasons and the values for the three m o n t h s of a season are pooled. The seasons are summer (April to June), p r e m o n s o o n (July to September), m o n s o o n (October to December) and postm o n s o o n (January to March). 4.1. Mugil cephalus 4.1.1. Skin and gill. F o r b o t h skin and gill the m i n i m u m count of luminous bacteria was rec o r d e d during the m o n s o o n period and the maxim u m during the p r e m o n s o o n season. The luminous microfloral count fluctuated from 1.3 • 104 to 3.6. 104 C F U / c m 2 in skin and 9 . 1 . 1 0 3 to 5 . 1 - 1 0 4 C F U / g wet wt. in gill. The luminous microbiota constituted 23.3-67.4% of the total heterotrophic bacterial population in skin, 21.8-88.4% of the same in gill (Table 1). L u m i n o u s microbes were n o t detected from the skin and gill in the m o n t h s of O c t o b e r and November. Species composition of the isolates revealed the occurrence of V. harveyi (78% in the skin, 92% in the gill) and V. fischeri (22% in the skin and 8% in the gill) (Fig. 1). 4.1.2. Gut. As observed in the case of the skin
40 2C 0 Gill
100
~ 8D "5 60
~o ~. 2o 0
lOC 80
60 z.O 20 0 A
M
J 1981
3
A
S MONTHS
0
N
O
J
F
M
]a82
Fig. 1. Species composition of luminous bacteria associated with skin, gill and gut of M. cephalus. I, V. harveyi; [], V. fischeri. Months are indicated by their first letters.
29
and the gill, the luminous bacterial load was also low in the gut during the monsoon season but a high bacterial load was noted during the postmonsoon season (Table 1). Within the gut, the foregut and midgut, but not the hindgut, showed minimum luminous counts during the monsoon season (Table 2). All the three gut regions showed maximum luminous bacterial population during the postmonsoon season. In the gut, the values for the luminous bacterial population ranged from 4.3-105 to 6 . 3 - 1 0 3 C F U / g dry weight of contents. Within the gut, the luminous CFU ranged from 2.1.104 to 5.1105 C F U / g dry wt. of contents in the foregut. In the midgut, the CFU ranged from 2.0.10 4 tO 3 . 5 - 1 0 5 / g dry wt. of contents, whereas in the hindgut the values fluctuated from 1.9-10 4 to 4.4" 105 C F U / g dry wt. of contents. As observed in the case of skin and gills the luminous microbiota was not detected in the gut region in October and November. The luminous CFU in the gut ranged from 13.2 to 79.3% of the total CFU (Table 1). In the three gut regions, the luminous microbial counts fluctuated from 26.0 to 74.6% in the foregut; from 17.7 to 87.0% in the midgut and 22.8 to 99.6% in the hindgut (Table 2). On an annual basis V. harveyi and V. fischeri accounted for 78.5 and 21.5% of the isolates, respectively, in the gut. Unlike V. fischeri, V.
Table 2 Percentage of luminous C F U a m o n g total C F U within the gut of Mugil cephalus Month
Foregut
Midgut
Hindgut
April 1981 May June July August September October November December January 1982 February March
57.7 27.3 61.2 26.0 0 47.6 0 0 1.6 69.1 74.6 59.2
18.7 17.7 87.0 31.1 0 53.8 0 0 47.8 74.6 67.3 67.5
53.0 22.8 57.6 62.7 40.2 89.3 0 0 99.6 69.0 82.0 47.0
harveyi was recorded continuously in all the regions of gut (Fig. 1). The quantitative distribution of luminous microflora was studied in relation to the type of food. A maximum luminous bacterial load was encountered during the postmonsoon season when algal filaments and diatoms dominated the diet. However, the same components with diatoms in lesser proportions were found in the diet of mullet (M. cephalus) during the monsoon season when the luminous bacterial load was also minimum. A negative relationship was observed between dry weight of the gut contents and luminous bacterial population in the gut (Fig. 2). 4.2. Tachysurus arius 4.2.1. Skin and gill. Minimum luminous bacterial count was recorded during the monsoon season, whereas the maximum was during the summer season. The luminous microfloral count ranged from 1.5.10 4 to 4.8.103 C F U / / c m 2 i n skin and 3.4.10 4 to 6.2" 1 0 3 C F U / g wet wt. in the gill. Skin and gill were devoid of luminous bacteria in October and November. The percentage composition of luminous microfloral population ranged from 8.4 to 84.3% of the total C F U on skin and 10.7 to 99.9% of the same in the gill (Table 3). On an annual basis V. harveyi constituted 84% of the luminous isolates on the skin and 82% in the gill, whereas V. fischeri accounted for 16% of the isolates in the skin and 18% in the gill (Fig. 3). 4.2.2. Gut. The luminous microfloral counts were less in the gut (in all the regions) during the monsoon season. But a high bacterial load was recorded during the postmonsoon season as in the case of skin and the gill (Tables 3 and 4). In the gut, the luminous microfloral counts varied from 2 .6 • 105 to 3.6.104 C F U / g dry wt. of contents of the three gut regions, the luminous bacterial count in the foregut ranging from 6.9.102 to 1.6-104 C F U / g dry wt. In the midgut, the count ranged from 3.4.104 to 3.7- 105 C F U / g dry wt. of gut contents, whereas in the hindgut the value varied from 3.2.104 to 5.3- 105 C F U / g dry wt. The luminous bacterial count in different regions of the gut ranged from 14.9 to 87.1% of the total bacterial count in the foregut, 8.6 to 88.6% in
30 6
Table 3
a
""-
5
/\z
\
Percentage of luminous CFU among total CFU associated with Tachysurus arius
.
Month
4
Region Skin
Gill
Gut
41.7 64.2 55.6 39.5 39.3 84.3 0 0 8.4 41.0 73.7 81.4
86.8 41.3 10.7 34.7 14.5 93.0 0 0 52.9 27.3 67.5 99.9
69.2 58.6 25.1 42.3 55.3 71.5 0 0 49.6 70.4 84.4 75.8
A
!
i
¢.o
::3
T ', I
3 ss
x
/
~
2
xA~
x
xg
s
"A
c
]
c
o 1:21.
b 0
i
t,._ II)
A
i
i
i
M J
i
J
A
i
i
i
S 0
'
l
i
N D J
J
F M
b z3
O t-
6
5
•\ / \ \
?=
o u
s "~ Z ._~
April 1981 May June July August September October November December January 1982 February March
./\ \.~"
•
t-"XD
""-"
.4
Gut
O
GI O
100
3
i i A,
"'~
t
80
i
~x II
i
b0
2 40 2C
1 a ,
A
i
i
M J
i
J
i
i
I
A S 0
i
i
i
N D J
i
F M
1982
1981 MONTHS
Fig. 2. Relationship between luminous bacterial population and dry weight of gut contents in M. cephalus (a) and T. arius (b). Here, gut bacterial populations were given as log CFU which is actually log of C F U / g dry weight of gut contents versus log of total dry weight of gut contents plotted against the sampling period. Months are indicated by their first letters.
8[
o: Skin ~13C
the midgut and 21.2 to 97.0% in the hindgut (Table 4). On an annual basis V. harveyi accounted for 76.8% of the isolates and II. fischeri 23.2% (Fig. 3). When gut content analysis of the fish was compared with the luminous bacterial load, the following trend was evident. Maximum luminous bacterial population was recorded in the postmonsoon season in T. arius when crustaceans dominated the diets. Minimum values were encountered during the monsoon season when fish,
8C
6{
Z.( 2
A
M
1981
J
J
A
S MONTHS
0
N
D
J
F
M
1982 Fi 9
"{
Fig. 3. Qualitative variation of luminous bacteria associated with skin, gill and gut of T. arius. Filled bars, V. haroeyi; hatched bars, V. fischeri. Months are indicated by their first letters.
31 Table 4 Percentage of luminous CFU among total CFU within the gut of Tachysurus arius Month
Foregut
Midgut
Hindgut
April 1981 May June July August September October November December January 1982 February March
65.1 58.0 77.9 76.7 14.9 57.4 0 0 39.5 69.6 82.6 87.1
64.8 37.7 8.6 8.6 88.1 82.4 0 0 45.5 88.6 71.8 64.3
82.3 21.2 90.4 76.7 74.4 83.5 0 0 63.4 72.3 97.0 78.3
polychaetes etc. formed the main food constituents. A negative relationship was observed between the weight of the food and luminous bacterial load in the gut (Fig. 2).
5. D I S C U S S I O N The three sites, namely the skin, the gill and the gut were selected in the two species of fish for studying the distribution and seasonal variation of the luminous microfloral population, because they offer ideal microenvironments [5] for the growth of these bacteria. The fishes M. cephalus and T. arius were chosen on the basis of their feeding habits. The former is a detritivore whereas the latter is a carnivore. They were selected to ascertain whether there is any relationship between the distribution of luminous procaryotes and the type of food consumed by these fishes. The extent of distribution of luminous bacteria a m o n g the total bacterial population (CFU) is quite interesting, 18-54% of the bacterial colonies isolated from the gut of Argyropelecus hemigymnus, Oxyjulis californica and Chromis punctipinnis being photogenic [6]. O'Brien and Sizemore [7] found that 0.44 and 20.55% of the total viable counts from stomach and intestine, respectively, were luminous. On the whole, the findings of the present investigation revealed that the luminous
microbiota (harboured in skin, gill and gut) constitute a significant portion of the total heterotrophic bacterial population with the luminous counts at times reaching up to 84% of the total counts. Maximum luminous counts were recorded in the skin of M. cephalus and T. arius during premonsoon and summer seasons, respectively, and minimum counts during the monsoon season. Generally, the skin of fishes is easily accessible to the bacterial flora of the surrounding water, and after colonization the bacteria thrive well on the slime secreted by the fish. The luminous bacterial density in the Vellar estuary was also correspondingly low (Table 5) during the monsoon period and hence a change in salinity of the water was reflected in the luminous bacterial count in the skin of the fish as well. The luminous microfloral population in the skin of M. cephalus was proportionately greater than that in T. arius. Since M. cephalus is a bottom feeder, the chances of this fish getting contaminated with sediment bacteria [8] cannot be ruled out and this may account for the higher incidence of microflora found on the skin. Horsley [9] was also of the opinion that fish inhabiting or in contact with the mud would get contaminated
Table 5 Water salinity and luminous bacterial population of water and sediment of the Vellar estuary Month
April 1981 May June July August September October November December January 1982 February March
Water salinity (%0)
Luminous CFU Water (CFU/ml)
Sediment ( C F U / g / d r y wt.)
32.3 30.6 31.8 21.6 30.5 32.0 17.0 18.0 21.7
31.5 33.1 18.5 13.5 6.7 19.9 6.4 3.5 3.5
32.4.103 27.3.103 24.3.103 16.9.103 17.4.103 10.2.103 51.0.102 50.6.10 2 85.1.102
29.4 28.9 29.2
25.1 24.1 31.3
16.8.103 12.5.103 23.1.103
32 from sediment microflora. In the present study, the maximum luminous bacterial load in the gill of M. cephalus was detected during the premonsoon season and in the gill of T. arius during the summer season. As observed in the case of skin, the minimum luminous bacterial density was recorded in the monsoon season in the gills of both fish. This can be ascribed to the depletion in luminous bacterial population in the surrounding medium during that period (Table 5). Since the gills are constantly exposed to the movement of water, any change in the bacterial density of the water should be reflected in the gill microfloral population also. Hence, it would be reasonable to assume that the low luminous bacterial population observed in the gill is indicative of the changes in the population size of the luminous bacteria in the water. Although salinity likely influences the size of the luminous bacterial population of the skin and the gill as a whole, minor variations in the numerical abundance of the luminous bacteria encountered could also be due to factors such as secretion of antimicrobial substances from the skin [10], tissue integrity, secretory bactericidins and production of mucus [11]. Data collected in the present study indicated that the luminous bacterial density of the gut in both the fishes (like that of the skin and gill) was influenced by seasonal changes. Since salinity seems to influence the luminous bacterial density in water (Table 5) it may indirectly influence the bacterial load of the gut also, because the fish secures its food from the water and sediment. Since M. cephalus and T. arius secure their food from the sediment and water, respectively, both of which recorded a very low luminous bacterial population in the monsoon months due to the effect of low salinity, it would be reasonable to infer that salinity indirectly influenced the bacterial load of the gut. Salinity-induced seasonal variations, similar to those in the present study, in the enteric luminous microflora of milk fish (Chanos chanos) have already been reported by Ramesh et al. [4]. The variations in the number of luminous bacteria in gut could be attributed to (a) the rate of emptying of the stomach which depends on food type, meal size, fish size and frequency of
feeding. Frequency of feeding in turn depends on texture, palpability of the food, digestibility and its energy and nutrient content [12]; (b) retention or residence time of food material in the gut [13] and (c) other factors such as previous feeding history of the animal [6] and its fatness and reproductive status [12]. These may also have a bearing on the gut microflora of the fish. Maximum luminous bacterial population was recorded in the alimentary tract of M. cephalus, in the postmonsoon season, when algal filaments and diatoms were the main constituents of the diet. Algal filaments are known to secrete metabolites which nourish the epiphytic microflora [14]. Lakshmanaperumalsamy and Purushothaman [15] reported the association of heterotrophic bacteria with seaweeds of the Vellar estuary. It is possible that, in M. cephalus, low-molecular-weight metabolites released from plankton and algae during the course of digestion could foster these bacterial populations. Though the same food components were encountered in the diet of mullet during the monsoon season also (when minimum luminous bacterial population was found), sand grains were greater in quantity. Maximum luminous bacterial load was registered in the gut of T. arius when crustaceans formed the major constituents of the diet. The association of luminous bacteria with crustaceans was documented by Baross et al. [16] and Sochard et al. [17]. Proliferation of luminous microbes might have taken place in the gut of T. arius as a result of the large amount of chitin available during that season. Hastings and Nealson [18] reported production of chitinase by luminous bacteria. Minimum counts were recorded when crustaceans were present in lesser proportions in the diet. Our observations concurred with those of O'Brien and Sizemore [7] who proved experimentally that fish fed with chitin-supplemented food registered greater counts of luminous bacteria than fish fed with food lacking chitin. Hence, the crustacean diet may account for the relative abundance of luminous bacteria in the gut of T. arius compared to that in M. cephalus. Attempts were also made to relate the luminous bacterial density in the gut of M. cephalus and T. arius to the weight of food present in the gut (Fig.
33 2). The negative relationship encountered suggested that the size of luminous microflora did not d e p e n d on the weight of the food alone. The findings of the present study are in contrast to those of R u b y and Morin [6] and O'Brien and Sizemore [7] who reported a relationship between the bacterial population and the feeding intensity of fish. It is also possible that the population size of the luminous microflora depends on the relative weight of various items in the diet rather than on the absolute weight of the food. The population density of luminous microbiota was greater in the gut than in the other microenvironments, viz., skin and gill, thus supporting the hypothesis of Horsley [9] that the fish gut acts as an enrichment vessel for the growth of bacteria. I n view of the extensive colonization of the digestive tract by the luminous procaryotes, the question naturally arises as to the possible function of these bacteria in the gut. The prime role of luminous bacteria in the gut of fish would appear to be nutritional, since all luminous procaryotes elaborate chitinase which degrades chitin in the gut into N-acetylglucosamine [19]. Peres et al. [20] f o u n d absorption o f N-acetylglucosamine in fish gut. V. haroeyi w a s the d o m i n a n t species in the gut, skin and gill of b o t h fishes. This was followed by V. fischeri. Preponderance of V. haroeyi in the gut was reported by Nair et al. [21], O'Brien and Sizemore [7] and R a m e s h et al. [22]. The main rationale for the distribution of V. harveyi might be its nutritional versatility [19] as it is able to utilize between 30 and 45 organic c o m p o u n d s (depending u p o n the strain). V. fischeri can utilise 7 - 2 2 and P. leiognathi only 15 c o m p o u n d s as nutrients. A n o t h e r plausible reason that could be advanced to explain the association of V. harveyi with fish is its flagellation. V. harveyi possesses lateral flagella, whereas polar flagellation is exhibited by V. fischeri and P. leiognathi. Belas and Colwell [23] studied the adsorption of laterally and polarly flagellated vibrios to chitin using the L a n g m u i r adsorption isotherm. T h e y reported a direct correlation between the lateral flagella production, surface binding nature of the flagella and the selective inhibition of polarly flagellated
bacteria by laterally flagellated bacteria. A l t h o u g h it was d e m o n s t r a t e d for V. parahaemolyticus, a species closely related to V. haroeyi, the same was likely to hold g o o d in the case of V. haroeyi, since its lateral flagella provide enough surface area for adsorption. McCall and Sizemore [24] screened a bacteriocin called ' h a r v e y c i n ' synthesized by V. harveyi which was k n o w n to be lethal to closely related bacteria. ~Bacteriocinogeny bestows a competitive advantage on V. harveyi to d o m i n a t e over nonbacteriocinogenic strains.
REFERENCES [1] Costerton, J.W. and Geesey, G.G. (1979) Microbial contamination of surfaces. In: Surface Contamination (Mittal, K.L., Ed.), Vol. I, pp. 211-221. Plenum Publishing Corporation, New York. [2] Potter, L.F. and Baker, G.E. (1961) The role of fish as conveyors of microorganisms in aquatic environments. Can. J. Microbiol. 7, 595-605. [3] Ruby, E.G. and Nealson, K.H. (1978) Seasonal changes in species composition of luminous bacteria in near shore seawater. Limnol. Oceanogr. 23, 530-533. [4] Ramesh, A., Nandakumar, R. and Venugopalan, V.K. (1986) Enteric luminous microflora of the pond-cultured mill
34
[13]
[14]
[15]
[16]
[17]
Control Processes in Fish Physiology (Ranklin, J.C., Pitcher, T.J. and Duggan, R.T., Eds.), pp. 23-40, Croom Helm Ltd., London. Hood, M.A. and Meyers, S.P. (1973) Microbial aspects of Penaeid shrimp digestion. Proc. Gulf and Caribbean fish. Inst., 26th Annual Session, October 1973, pp. 81-92. Fogg, G.E. (1966) Extracellular products of algae. In: Oceanogr. Mar. Biol. Annu. Rev. (Barnes, H., Ed.), pp. 195-212. George Allen and Unwin Ltd., London. Lakshmanaperumalsamy, P. and Purushothaman, A. (1982) Heterotrophic bacteria associated with seaweed. Proc. Indian Acad. Sci. (Plant Sei). 91,487-493. Baross, J.A., Tester, P.A. and Morita, R.Y. (1978) Incidence, microscopy and etiology of exoskeleton lesions in the tanner crab, Chinoecetes tanneri. J. Fish. Res. Bd. Can. 35, 1141-1149. Sochard, M.R., Wilson, D.F., Austin, B. and Colwell, R.R. (1979) Bacteria associated with the surface and gut of marine copepods. Appl. Environ. Microbiol. 37, 750-759.
[18] Hastings, J.W. and Nealson, K.H. (1977) Bacterial bioluminescence. Annu. Rev. Microbiol. 31, 549-595. [19] Reichelt, J.L. and Baumann, P. (1973) Taxonomy of the marine luminous bacteria. Arch. Microbioi. 94, 283-330. [20] Peres, G., Boge, B., Colin, D. and Rigal, A. (1973) Effects of temperature on the fish digestive process. Enzymic activities and intestinal absorption. Rev. Trab. Inst. (Sci. Yech.) Pech. Marit. 37, 223-232. [21] Nair, G.B., Abraham, M. and Natarajan, R. (1979) Isolation and identification of luminous bacteria from Porto Novo estuarine environs. Indian J. Mar. Sci. 8, 46-48. [22] Ramesh, A., Loganathan, B. and Venugopalan, V.K. (1985) Enteric luminous bacteria of bivalves from Porto Novo waters. J. Singapore Nat. Acad. Sci. 14, 116-119. [23] Belas, M.R. and Colwell, R.R. (1982) Adsorption kinetics of laterally and polarly flagellated Vibrio. J. Bacteriol. 151, 1568-1580. [24] McCall, J.O. and Sizemore, R.K. (1979) Description of a bacteriocinogenic plasmid in Beneckea harveyi Appl. Environ. Microbiol. 38, 974-979.