Microbiology of summer flounder Paralichthys dentatus fingerling production at a marine fish hatchery

Aquaculture 211 (2002) 9 – 28 www.elsevier.com/locate/aqua-online

Microbiology of summer flounder Paralichthys dentatus fingerling production at a marine fish hatchery Stephen D. Eddy a,1, Stephen H. Jones b,* b

a GreatBay Aquafarms, 153 Gosling Road, Portsmouth, NH, USA Jackson Estuarine Laboratory, University of New Hampshire, 85 Adams Point Road, Durham, NH 03824-3427, USA

Received 1 July 2001; received in revised form 15 November 2001; accepted 18 November 2001

Abstract The microbiology of summer flounder, Paralichthys dentatus, fingerling production was monitored over the course of 10 production cycles during 1996 – 1999 at a commercial marine fish hatchery. Samples of the rearing water, fish larvae and live feed were analyzed to quantify the total heterotrophic bacteria (marine agar) and total presumptive vibrios. Selected bacterial isolates were characterized to the group or species level. The tank water was compared between two larviculture methods to see if greenwater densities or the timing of fish movements could affect the tank microbiology. The extensive use of phytoplankton as rotifer enrichment and as greenwater reduced the incidence of vibrios as a percentage of total heterotrophs in the rotifers and water. Rinsed enriched Artemia had high levels of vibrios, and the fish larvae experienced increased mortality during the period of Artemia feeding although known bacterial fish pathogens were not detected at significant levels. The microbiota of the rearing water and fish intestine were similar to that of the live feed being utilized at the time of the sampling. A succession of bacterial phenotypes was observed in the rearing water and the fish intestine from day 1 to day 90 post-hatch, and the fish larvae showed evidence of the development of a stable indigenous microbiota during and after metamorphosis. Acinetobacter, Agrobacterium, Flavobacterium, Moraxella and Pseudomonas were the dominant bacterial groups in phytoplankton, rotifers and the early larval fish and tanks. However, Artemia and older larval stages and tanks showed a shift towards the microbiota with higher levels of Vibrio and Enterobacter. Bacillus was detected in juvenile fish but not in larval stages. The results provide a database for analyzing the role of the microbiota in health and disease

* Corresponding author. Tel.: +1-603-862-2175; fax: +1-603-862-1101. E-mail address: [email protected] (S.H. Jones). 1 Present address: Center for Cooperative Aquaculture Research, 33 S. Bay Road, Franklin, ME 04634, USA. 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 8 2 - 1

10

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

of summer flounder and suggest that this microbiota may be amenable to probiotic type manipulations. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Microbiology; Microbial monitoring; Vibrio index; Probiotics; Paralichthys dentatus; Summer flounder; Larval rearing

1. Introduction Economically viable finfish mariculture requires a reliable and plentiful supply of healthy fingerling fish, which in many cases are produced using intensive hatchery techniques. A significant factor affecting the outcome of hatchery fingerling production is the microbiology of the fish-rearing biotope. Opportunistic bacterial pathogens, particularly Vibrio spp., are present as part of the normal microbiota of marine fish and have been shown to be causative agents of disease and mass mortality (Horne et al., 1977; Muroga et al., 1990; Grisez et al., 1996; Sedano et al., 1996; Pedersen et al., 1997). Larviculture tanks used in intensive production can become high in organic matter, providing substrates for microbial growth and leading to the proliferation of fast-growing opportunistic bacteria (Hansen and Olafsen, 1989; Skjermo and Vadstein, 1999). High microbial numbers associated with live feed and/or the fish’s rearing environment can result in poor survival and performance of the larvae (Muroga et al., 1987; Nicolas et al., 1989; Munro et al., 1994). Chemotherapy is commonly used for microbial control in aquaculture but can lead to the development of drug-resistant strains (Riquelme et al., 1995; Kapetenaki et al., 1995; Hameed and Balasubramanian, 2000) and may delay or inhibit the colonization of the fish and its environment by non-pathogenic or potentially beneficial bacteria (Austin and AlZahrani, 1988; Hansen et al., 1992). Alternatives to chemotherapy include the use of ‘microbially matured’ rearing water colonized by non-opportunistic bacteria (Skjermo et al., 1997; Skjermo and Vadstein, 1999), biocontrol using autochthonous microorganisms to repress the growth of pathogens in the rearing environment (Nogami and Maeda, 1992; Maeda, 1994; Riquelme et al., 1997), and probiotic bacteria to exclude or inhibit pathogens from colonizing fish (Smith and Davey, 1993; Gatesoupe, 1994; Austin et al., 1995; Sakai et al., 1995; Gildberg et al., 1997; Gram et al., 1999). These approaches require the determination of the microbial community and ecology associated with a species and the culture system within which it is reared (Nicolas et al., 1989; Olsson et al., 1992; Munro et al., 1995a,b; Kennedy et al., 1998; Gatesoupe, 1999). This aids in identifying pathogens and other bacteria associated with success or failure and can ultimately lead to the selection of probiotic microbial isolates and communities. Summer flounder, Paralichthys dentatus, is a recently commercialized fish grown in the northeast United States (Bengston, 1999; Bengston and Nardi, 2000). The microbial monitoring of summer flounder fingerling production at a commercial hatchery was begun in 1996 as part of a larger effort to optimize the health and microbiology of this species in culture. The preliminary objective was to establish baseline microbiological data, which could then be used to further examine issues of health and disease in fingerling production

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

11

and evaluate alternative strategies for microbial control. Selective and non-selective media were used to estimate numbers and obtain isolates of heterotrophs and Vibrio spp. associated with fish, feed and water at different developmental stages. Isolates were characterized to identify the numerically dominant bacterial groups and Vibrio spp. Microbial levels and composition in tank water were compared between tanks under two larviculture regimes utilizing low and high greenwater densities of the marine algae Nannochloropsis oculata. Findings relating to the general microbiology of summer flounder fingerling production were then examined in the context of similar previously published studies on other marine fish.

2. Materials and methods The study was performed at GreatBay Aquafarms in Portsmouth, NH and at the Jackson Estuarine Laboratory, University of New Hampshire, Durham, NH. 2.1. Larviculture Techniques for summer flounder broodstock maintenance and conditioning, spawning and egg incubation have been described (Watanabe et al., 1998; Bengston, 1999). Newly fertilized eggs were disinfected for 10 min in 50 mg/l providone iodine before incubation. Two greenwater-rearing methods, described as low-density and high-density, were utilized for the larviculture. All phytoplankton and zooplankton used for the larval feeds were grown and enriched on the site using standard methods (Hoff and Snell, 1993; Baker et al., 1998). In low-density larviculture, cohorts of newly hatched larvae were stocked and raised in 4.5-m3 round tanks for 40 – 60 days at densities of 20 larvae/l. Greenwater levels of N. oculata were maintained in the rearing tanks for the first 12 days post hatch (DPH) at tank densities of approximately 104 cells/ml as determined using a Hach spectrophotometer (Hach DR/2000). Larvae were fed rotifers Brachionus plicatilis enriched for 4 –12 h in a mix of high density living phytoplankton consisting of Isochrysis sp., Tetraselmis sp. and Nannochloropsis sp. Feedings took place at frequent intervals on a daily basis. At 15 –17 DPH, the larvae were transitioned to nauplii of the brine shrimp A. salinas for a period of 2 –4 days, after which they were fed exclusively on Artemia enriched in DHA Selco (Inve) until approximately 45– 50 DPH. Weaning to dry diets was commenced at this time, and the metamorphosed larvae were transferred at 45 – 60 DPH to smaller (1 m3) square weaning tanks for the final complete transition to artificial feeds. In high-density larviculture, flounder eggs were stocked just prior to hatch into 1-m3 round tanks at densities of 60 –80 larvae/l for in-tank hatching and early larval rearing. Greenwater levels of N. oculata were maintained for the first 10– 12 DPH at densities of approximately 107 cells/ml as evaluated by the Secchi disk and Hach spectrophotometer. An initial small inoculation of rotifers resulted in a population bloom within the tanks as they grazed upon the algae, so daily additions of enriched rotifers were not required. At 14 –17 DPH, the larvae were transferred to larger tanks (4.5 m3) for rearing at lower densities. Artemia feeding and weaning took place in these larger tanks.

12

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Four distinct developmental and feeding stages were differentiated for larval and fingerling summer flounder by age group given as days post hatch (DPH) and described as stage 1 (early larval, 1 – 15 DPH), stage 2 (mid-larval, 16 – 29 DPH), stage 3 (metamorphosing, 30– 49 DPH) and stage 4 (juvenile, 50 – 95 DPH). Stage 1 fish were 3.4 –7.8 mm, had a straight undifferentiated gut and fed exclusively upon rotifers. Stage 2 fish were 7.0– 11.4 mm, showed early gut differentiation characterized by a single coil and were fed Artemia and/or early weaning dry diets. Stage 3 fish were 11.0 –17.4 mm, undergoing metamorphoses and were fed enriched Artemia and/or dry diets. Stage 4 summer flounder were 15– 30 mm, fully metamorphosed with a differentiated gut and were fed only on dry diets. 2.2. Bacteriological methods Samples were collected from 10 consecutive production cycles during 1996– 1999, with each cycle consisting of a cohort of fish of mixed parentage spawned within a 1 –2week time period and raised as a batch in a group of tanks to a fingerling size of 5– 10 cm. Sampling frequency was weekly or at transitions such as changeover from one feed type to another, metamorphosis and movement to new tanks. All samples were collected in autoclaved sterile 1-l polypropylene jars. Samples of phytoplankton and rotifers were collected directly from culture tanks and/or just prior to being used as greenwater or feed. Fish eggs, larval fish and tank water samples were also collected directly from culture tanks. Artemia were rinsed to reduce their microbial load by harvesting into 160-Am mesh bags and rinsing for 15 min in freshwater. They were then re-suspended in 1-Am filtered UV-sterilized seawater at a density of 1000/ml before being used as feed or sampled for microbiological analyses. Artemia densities were determined using five replicate counts using 1-ml subsamples. Samples were transported on ice to the Jackson Estuarine Laboratory at the University of New Hampshire and processed within 2 h of collection. Water, algae and rotifers were serially diluted with no pre-treatment in sterile seawater or buffered peptone water. Artemia and fish eggs were homogenized along with their culture water in sterile 15-ml glass tissue grinders (Wheaton, USA) before serial dilution. Fish were lethally anesthetized with MS222 before being processed. Early larval, mid-larval and metamorphosing fish were pooled as tank groups (2 –10 fish per group), and larger juvenile fish were treated as individuals. Microbial sampling of fish was designed primarily to select for gut bacteria. The fish were surface sterilized with a 30-s immersion in 0.1% benzalkonium chloride followed by a 30-s rinse in sterile distilled water using the methods of Muroga et al. (1987). They were then homogenized with glass tissue grinders in sterile seawater to a total volume of 10 ml, vortexed to thoroughly mix and serially diluted as for the other samples. All samples were plated as follows. Ten-fold dilutions of samples were filtered through 0.45-Am membrane filters (Gelman) which were plated on thiosulfate citrate bile salt sucrose (TCBS, Difco) (Bolinches et al., 1988) and V. anguillarum medium (VAM, Difco) (Alsina et al., 1994) agar for the selective isolation and quantification of Vibrio spp. and V. anguillarum, respectively. Each dilution was also plated as 100 Al on replicate 2216 marine agar (Difco) plates for the quantification of culturable heterotrophs. Plates were incubated at room temperature (18 – 22 jC) for 24 – 72 h until the colonies were established. Total colonies and colony types were enumerated on plates with 20– 200

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

13

colonies to obtain colony-forming units (CFU), and individual colonies were picked for isolation and characterization. Criteria for isolation included rarity or unusual appearance as well as abundance, but no more than six colony types from a single plate were isolated. Isolates were frozen at 80 jC in freeze media comprised of brain – heart infusion broth (BHI) (Difco) supplemented with 1.0% NaCl and 15% glycerine. All strains identified and used in this study were maintained on tryptic soy agar (TSA) (Difco) or Luria agar (Difco) supplemented with 1.5% NaCl. Isolate characterization was done to identify the dominant groups of bacteria. A total of 1109 isolates were either partially or completely characterized using biochemical tests. The bacteria were grouped according to the scheme devised by Muroga et al. (1987) in order to classify bacteria to the genus level. In many cases, members of the genus Vibrio were further classified to the group and species level in order to identify the dominant groups and the occurrence of known vibrio fish pathogens, following a simplified scheme based on West et al. (1986), Alsina and Blanch (1994) and McLaughlin (1995). The media used for the characterization were prepared using standard formulations or were purchased as commercially available tests. All prepared media were supplemented with 1.5% NaCl. The prepared media included all salinity tests, arabinose, cellobiose, lactose and salicin carbohydrate tests, Hugh – Leifson O/F media, urease, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and motility media. VAM agar used for the selective growth of Vibrio spp. and for testing the acid production from sorbitol was prepared according to Alsina et al. (1994). TCBS agar was used for the selective growth of Vibrio spp. and to test for acid from sucrose. Blood agar was prepared by the University of New Hampshire Veterinary Diagnostics Laboratory using TSA and sheep blood erythrocytes. Commercial test products used included oxidase (Difco), catalase (Difco), Gram stain kit (Difco) and indole (Difco). 2.3. Data analyses Samples taken throughout the course of the study were pooled for data analyses by type and fish developmental stage. In addition, samples of tank water were compared at each of the first three developmental stages between the two larviculture methods (high-density larviculture vs. low-density larviculture). The geometric mean of the bacterial counts quantified on TCBS and marine agar was calculated for each pooled sample grouping. A vibrio index was calculated for each sample grouping as a proportion equal to the geometric mean TCBS CFU (total presumptive vibrios)/the geometric mean marine agar CFU (total culturable heterotrophs). The vibrio proportion index, geometric means on TCBS and marine agar and the maximum CFU/ml or CFU/fish observed on marine agar were then used to compare the sample groups and to look for gross trends in microbial numbers and communities.

3. Results The geometric mean bacterial counts, given as CFU/ml or CFU/fish on TCBS and marine agar, and the maximum observed CFU/ml or fish on marine agar are presented for

14

Bacterial group

Motility media

Hugh – Leifson/ oxidative

Hugh – Leifson/ fermentative

Oxidase

Catalase

Growth on TCBS agar

Other characteristics

Acinetobacter Aeromonas

+

+

+

+

+ +

+ p

Agrobacterium Alcaligenes Enterobacter

+ + v

+

+ + +

v +

v

p

+ +

+ +

+ p

+

+

+

Growth on MacConkey agar Usually ferments sucrose; gas from glucose Copious slime on carbohydrates Limited action on carbohydrates Gas often produced during fermentation Yellow or orange pigmented colonies No acid from carbohydrates Growth on Cetrimide agar; salt media not required Most require NaCl for growth

+

Flavobacterium/ Cytophaga Moraxella Pseudomonas

+

v

Vibrio

+

+

v: Variable results; p: poor growth.

+ + +

+

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Table 1 Growth characteristics of major Gram-negative bacterial groups (Muroga et al., 1987) identified at GreatBay Aquafarms

Table 2 Biochemical characteristics of major vibrio groups and species (West et al., 1986; Alsina and Blanch, 1994; McLaughlin, 1995) identified at GreatBay Aquafarms Vibrio group or species

Group I vibrios V. natriegens V. ordalii V. pelagious Group II vibrios V. anguillarum V. fluvialis V. furnissii Group III vibrios V. fischerii Group IV vibrios Photobacterium damselae Group V vibrios V. alginolyticus V. carchariae V. parahaemolyticus V. vulnificus Group VI vibrios Group VII vibrios +

+ + + + + + + + + + + + + + + + + + +

v v

v v

+ v v v + v

+ + + +

v v

+ +

+ + + +

+ v

+ + + + + + v

v + v v v

v + v + v v

+ + + + + + v

v

Acid Acid Acid Acid Acid Hemolysis Indole Urease from from from on blood formation from from cello- lactose salicin sorbitol sucrose biose v +

v

v

v + v

v + + + v + + + v

v

v

v +

v

v +

v v +

v

v

v

v

v

v

v

+

v + +

+ v v

v v

v v

+ + + v v

+ v

+ v v

+ + v

+ v + + v + v +

v

v

v +

v

v

v +

v v

+ v v

+

v

v v

v

v v

v v v

v

v

v

v

+

v

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Growth Growth Growth Arginine Lysine Ornithine Acid at 0% at 3% at 8% dihydro- decarbo- decarbo- from NaCl NaCl NaCl lase xylase xylase arabinose

nd nd

v: Variable results; nd: not done.

15

16

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

all sample groups in Fig. 1. The vibrio proportion index for each sample group is presented in Fig. 2. Growth and biochemical test results for classifying and grouping isolates are summarized in Tables 1 and 2. Occurrences of major bacterial groups and maximum CFU/ ml or CFU/fish observed for each group are summarized in Table 3 for water samples, Table 4 for live feed samples and Table 5 for fish samples. 3.1. Water samples The supply water used at GreatBay Aquafarms had total bacterial plate counts on marine agar ranging from 7 to 15,000 CFU/ml. Corresponding plate counts on TCBS were negligible, and the characterization of the isolates picked from marine agar indicated that 80– 90% of the total incoming bacteria were non-vibrios. Tank water associated with high-density and low-density larviculture of stage 1 larvae (1– 15 DPH) showed low levels of presumptive vibrios and consequently had a low vibrio index (Figs. 1 and 2). High-density tanks had higher levels of total heterotrophs (about 105 CFU/ml, marine agar) than did low-density tanks (about 104 CFU/ml on marine agar). Numerically dominant isolates found in early larval tank water were similar to those seen

Table 3 Occurrences of major bacterial groups and maximum CFU/ml observed in supply water and tank water at GreatBay Aquafarms: 1996 – 1999 Bacterial type

Acinetobacter spp. Agrobacterium spp. Alcaligenes spp. Enterobacter/ Pasteurella/ Aeromonas spp. Flavobacterium/ Cytophaga spp. Moraxella spp. Pseudomonas spp. Vibrio spp. Gram-positive cocci Unidentified

Supply water

Stage 1 water

Stage 2 water

Stage 3 water

Stage 4 water

Total number of samples

7

28

22

12

20

Total number of isolates

32

182

152

67

96

Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU

3 1.3  101 0

5 7.2  104 2 2.8  105 3 1.2  105 3 2.8  104

4 7.2  104 1 8.4  104 4 1.0  105 2 2.8  104

3 5.0  104 3 2.8  105 3 2.8  104 2 2.3  105

2 8.8  105 1 5.0  103 5 2.6  105 2 8.0  102

Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates

4 6.0  102 8 0.3  101 0

8 4.0  104 12 3.2  105 0

2 2.4  106 3 1.3  106 0

9 1.8  103 0

9 2.5  104 19 3.8  105 2 2.0  103 105 7.0  104 0

105 6.0  105 0

47 2.5  106 0

4

34

16

4

1 2.8  102 6 3.0  105 1 4.0  103 51 3.9  105 1 8.0  103 26

2 0.1  101 2 0.1  101

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Fig. 1. Maximum and mean CFU/ml or CFU/fish for all sample groups: 1996 – 1999.

17

18

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

associated with phytoplankton and included Alcaligenes spp. and Moraxella spp. (Table 3). Acinetobacter spp. and Flavobacterium/Cytophaga spp. were also frequently isolated but at lower levels. At stage 2 (16 – 29 DPH), the total bacterial counts of the tank water increased to 2.4  105 in high-density tanks but remained similar to stage 1 levels in low-density larviculture tanks at 3.1  104 (Fig. 1). The vibrio index increased under both larviculture regimes but was substantially higher in low-density larviculture tanks (0.5) than in highdensity larviculture tanks (0.04) (Fig. 2).

Fig. 2. Vibrio index calculated for all sample groups as a proportion equal to the geometric mean TCBS CFU/the geometric mean marine agar CFU.

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

19

During stage 3 (30 –49 DPH), the mean and maximum bacterial plate counts of tank water did not differ substantially between the larviculture regimes, so the data for these samples were combined for the analyses. Mean and maximum tank bacterial levels increased during stage 3 to 1.5  106 and 4.2  106 CFU/ml, respectively (Fig. 1). The vibrio index remained high but was similar for both high-density and low-density larviculture tanks (0.24), showing a decline from the high levels seen in stage 2 lowdensity larviculture tanks (Fig. 2). Total bacterial counts for stage 4 (50 –95 DPH) tanks were not differentiated on the basis of larviculture method. Mean bacterial counts of the stage 4 tank water as quantified on marine agar were 105 – 106 CFU/ml (Fig. 1). The vibrio index for stage 4 tanks dropped to 0.03, showing a substantial decline from levels seen in the tanks used for earlier life stages (Fig. 1). Stage 4 rearing tanks had high species diversity, and numerically dominant heterotrophs included Acinetobacter spp., Alcaligenes spp. and Moraxella spp. (Table 3). A Bacillus sp. was detected on one occasion in stage 4 tank water at 8.0  103 CFU/ml. This was one of the few occasions during the course of this study when a Gram-positive microbe was detected.

3.2. Live feed samples Total bacterial levels quantified on marine agar for phytoplankton cultures averaged 3.3  105 CFU/ml although a maximum of 1.1  107 CFU/ml was observed (Fig. 1). No obvious differences were detected in bacterial numbers or groups between the three species of algae grown in mass culture (data not shown). The vibrio index for all algal samples was consistently low at 0.0001 (Fig. 2), and the bacterial counts on TCBS averaged only 23 CFU/ml (Fig. 1). Rotifers from stock culture tanks that were primarily fed with yeast had high vibrio levels of up to 105 CFU/ml on TCBS (data not shown), but after a 12-h algal enrichment, the vibrios were barely detectable, resulting in a low vibrio index of 0.001 for rotifers used as feed (Figs. 1 and 2). Mean total heterotrophic bacterial counts for enriched rotifers were about 106 CFU/ml, similar to levels seen for phytoplankton. Numerically dominant heterotrophs found in algal-enriched rotifers included Moraxella spp., Acinetobacter spp. and Flavobacterium/Cytophaga spp. Rotifers from stock tanks and algal-enriched rotifers also had isolates rarely found to be associated with the phytoplankton, such as Pseudomonas spp. and Enterobacter spp. (Table 4). The Artemia enrichment protocol used chlorine-sterilized seawater as a culture medium, but after 24 h, the enriched Artemia that was harvested and rinsed as previously described had as many as 1.4  107 CFU/ml quantified on marine agar (Fig. 1). Characterization of these colonies showed that on some occasions, up to 80% of the bacteria associated with Artemia were vibrios, with the overall result showing that Artemia had a much higher vibrio index (0.2) than phytoplankton or rotifers (Fig. 2). V. alginolyticus was a frequent isolate that was found to be associated with Artemia and was often detected at high levels (about 106 CFU/ml). The bacteria from the Enterobacter/ Pasteurella/Aeromonas grouping were also frequently encountered at levels of up to 3.0  106 (Table 4).

20

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Table 4 Occurrences of major bacterial groups and maximum CFU/ml observed in plankton feeds at GreatBay Aquafarms: 1996 – 1999 Bacterial type

Acinetobacter spp. Agrobacterium spp. Alcaligenes spp. Enterobacter/Pasteurella/ Aeromonas spp. Flavobacterium/ Cytophaga spp. Moraxella spp. Pseudomonas spp. Vibrio spp. Gram-positive cocci Unidentified

Phytoplankton

Rotifers

Artemia

Total number of samples

15

11

9

Total number of isolates

52

74

60

Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Number of isolates

5 8.3  105 0

2 2.7  106 1 4.0  101 3 8.3  105 6 4.7  105 3 4.0  103 9 2.2  106 5 1.2  103 39 5.0  105 0 6

1 8.0  102 0

3 6.7  104 1 4.9  104 4 3.3  105 16 1.0  107 2 8.3  104 14 3.4  103 0 7

0 5 3.0  106 0 1 1.0  103 0 52 2.4  106 0 1

3.3. Eggs and fish After 2 days of incubation at 18 jC, the total bacterial levels of summer flounder eggs and associated hatch water, as quantified with marine agar, had a geometric mean of 6.6  106 CFU/ml. Bacterial counts obtained with TCBS were highly variable and ranged from 101 to 107 CFU/ml, with a geometric mean of 1.2  104 CFU/ml (Fig. 1). Overall, summer flounder eggs had a low vibrio index of 0.01, and Moraxella spp. was the numerically dominant heterotroph. Early larval stage 1 summer flounder had low levels of intestinal bacteria per fish, averaging 101 CFU/fish on TCBS and 1.3  103 CFU/fish on marine agar (Fig. 1). Numerically dominant isolates included Moraxella spp. and Pseudomonas spp. (Table 5). Although the vibrios were frequently isolated, they were present at trace levels and the early larval fish consequently had a low vibrio index of 0.008 (Fig. 2). Stage 2 fish had total heterotroph counts of 4.7  104 CFU/fish on marine agar, with a maximum observed level of 1.3  105 CFU/fish (Fig. 1). Characterization of bacterial isolates from stage 2 fish showed that on some occasions, up to 80% of the isolates were vibrios. Stage 2 fish had the highest vibrio index (0.5) observed for all fish stages (Fig. 2), with average bacterial counts on TCBS of 2.3  104 CFU/fish. In particular, V. alginolyticus was identified in 57% of the sampled stage 2 fish. Non-vibrio bacteria identified in these larvae were predominately Acinetobacter spp., Enterobacter spp. and Moraxella spp. Flavobacterium/Cytophaga spp. seen in early larval stage 1 fish were not detected in stage 2 fish or in subsequent developmental stages (Table 5).

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

21

Table 5 Occurrences of major bacterial groups and maximum CFU/fish observed in different summer flounder developmental stages at GreatBay Aquafarms: 1996 – 1999 Bacterial type

Acinetobacter spp. Agrobacterium spp. Alcaligenes spp. Enterobacter/ Pasteurella/ Aeromonas spp. Flavobacterium/ Cytophaga spp. Moraxella spp. Pseudomonas spp. Vibrio spp. Gram-positive cocci Unidentified

Stage 1 larval fish (1 – 15 DPH)

Stage 2 larval fish (16 – 29 DPH)

Stage 3 larval fish (30 – 49 DPH)

Stage 4 juvenile fish (50 – 94 DPH)

Total number of samples

8

10

12

31

Total number of isolates

28

61

98

190

Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU

1 1.4  103 1 2.7  103 2 8.0  102 0

3 1.4  105 1 2.0  104 1 4.0  103 4 7.0  104

5 4.2  104 0

2 6.3  104 6 1.1  106 4 1.4  107 0

Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates Maximum CFU Number of isolates

2 2.2  103 2 1.7  104 2 4.1  104 16 2.6  102 0

0

0

0

2 8.0  102 0 46 1.1  105 0

3 1.1  105 1 ND 77 4.2  106 0

2

4

6

1 ND 3 5.0  104 146 2.3  106 2 4.0  103 26

2 1.5  102 6 8.0  103

Metamorphosing (stage 3) summer flounder had intestinal bacterial levels averaging 3.9  104 CFU/fish as quantified on marine agar. Numerically abundant non-vibrio heterotrophs were Acinetobacter spp., Moraxella spp. and Enterobacter spp. (Table 5) although the vibrios remained a significant and often dominant component of the microbial population (vibrio index = 0.16) (Fig. 2). Most of the vibrio isolates were phenotypically similar to those seen in earlier life stages of the fish (such as V. alginolyticus), but isolates identified as V. fluvialis and V. fischerii were detected in 25% of the larvae, compared to being detected in only 6% of the earlier stage larvae (Table 2). Total bacterial numbers increased in juvenile (stage 4) fish, averaging 1.3  105 CFU/ fish on marine agar (Fig. 1). The total bacteria quantified on TCBS also increased to an average of 1.0  104 CFU/fish, but the vibrio index showed a decline of 0.08 from the previous levels (Fig. 2). The species composition of juvenile fish was more varied and differed from the earlier life stages. V. alginolyticus was not detected in juvenile fish, but V. fluvialis and V. fischerii were identified in 38% of the sampled juvenile fish, with V. fischerii detected at the highest numbers (2.3  106 CFU/fish) seen for vibrio isolates in all sampled fish. Non-vibrio isolates identified as Agrobacterium spp. and Alcaligenes spp. were sometimes enumerated at high levels in juvenile fish (Table 5). In addition, the Gram-

22

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

positive cocci identified as a Bacillus sp. were detected in two juvenile fish at levels of approximately 103 CFU/fish.

4. Discussion Supply water can be a major source of the microbes associated with an aquaculture facility (Douillet and Pickering, 1999). In the present study, some of the bacterial isolates found in the live feed, fish intestine and tank water were not detected in the supply water (Table 1). These microbes may have originated in ‘starter’ plankton cultures or have been introduced by humans and broodstock fish. Regardless of the origin, a successional pattern was observed in the tank water and fish intestine that appeared to be closely correlated with live feed inputs. Tank water microbiology differed quantitatively and qualitatively at different stages of summer flounder larval development. The differences were most dramatic during the first two larval stages between the two rearing methods. Higher bacterial levels seen in highdensity tanks as compared to low-density tanks during stage 1 may be due to the different algal densities maintained under the two rearing regimes. Phytoplankton comprised up to 60% of the total tank volume in high-density tanks vs. approximately 4% of the tank volume in low-density tanks. Bacterial populations co-existing with the algae were on the magnitude of 106 CFU/ml. Thus, the numerically dominant heterotrophs associated with phytoplankton also dominated early larval tank water. Differences in tank water microbiology persisted between the two larviculture regimes through stage 2 after the greenwater had been discontinued. Stage 2 high-density tanks had higher bacterial levels than stage 2 low-density tanks but had lower levels of Vibrio spp. (Fig. 2). In contrast, stage 2 low-density tank water had the highest vibrio levels seen for any larval stage. These differences may in part be attributable to the fact that during the high-density culture, newly cleaned tanks were used for stage 2, whereas in low-density culture, stage 2 larvae were kept in the same tanks. Verschuere et al. (1997) and Skjermo and Vadstein (1999) theorized that in an aquaculture tank with low levels of organic substrate, slowly growing non-opportunistic bacteria (‘K-strategists’) are favored over fast-growing opportunistic ‘r-strategists.’ Many Vibrio species, particularly those associated with Artemia, are r-strategists (Verschuere et al., 1997). Higher levels of Artemia-associated vibrios in stage 2 low-density larviculture tanks may be due to the higher organic substrate levels present in these ‘‘older’’ and ‘‘dirtier’’ tanks. Counterbalancing this tendency in a mature microbial ecosystem, such as that found in an older tank, can favor K-strategists because it is a crowded environment with low substrate supply per bacterium (Andrews and Harris, 1986). Skjermo et al. (1997) used ‘microbial maturation’ of tank water to promote a microbial community dominated by K-strategists and low in vibrio pathogens. In addition, although the organic matter content may be higher in older fish tanks, it can also be recalcitrant to decomposition, making it less readily available as the organic growth substrate. However, continuous inputs of Artemia, combined with tank bottom cleanings, which tended to stir up debris, may have prevented the formation of a ‘mature’ and stable bacterial community in stage 2 low-density tanks.

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

23

Evaluating the effect of tank water microbial composition upon fish intestine microbial composition was difficult due to the complicating variable of feed input. It is generally accepted that the microbiota associated with non-feeding early larval fish is transient and strongly influenced by the water (Campbell and Buswell, 1983; Munro et al., 1994; Ringo et al., 1996; Ringo and Birkbeck, 1999). In summer flounder eggs and yolk-sac larvae, vibrios were a minor component of the microbial community, and Moraxella sp. was the numerically dominant heterotroph. This genus was also found to be associated with turbot eggs by Keskin et al. (1994), and similar findings have been reported by others (Hansen and Olafsen, 1999). With the onset of first feeding, the intestinal microbiota of cultured marine fish becomes increasingly dominated by the bacteria associated with the live feed (Muroga et al., 1987; Munro et al., 1994; Grisez et al., 1997). In particular, the phytoplankton used as greenwater and as a live feed enrichment can considerably alter the gut microbiota of larval fish (Nicolas et al., 1989; Bergh et al., 1994; Reitan et al., 1997; Olsen et al., 2000). Reitan et al. (1997) speculated that in addition to the known nutritional benefits, phytoplankton has microbiological benefits. Rico-Mora et al. (1998) found that the bacteria associated with the alga Skeletonema costatum were able to competitively exclude V. alginolyticus and proposed that the microalgae be used as a low-cost source of probiotic bacteria. Austin et al. (1992) showed that T. suecica inhibited the growth of bacterial pathogens including vibrios. The present study suggests that phytoplankton strongly influenced the microbiology of the stage 1 rearing environment. Newly feeding summer flounder, rotifers and tank water were all shown to be dominated by the same non-vibrio heterotrophs found in the phytoplankton used for greenwater and enrichments. This contrasts with Munro et al. (1994), who found that the gut of the 5-day larval turbot Scophthalmus maximus feeding on algal-enriched rotifers was dominated by vibrios at levels of approximately 105 CFU/fish. Bergh et al. (1994) showed that larval halibut Hippoglossus hippoglossus underwent a shift in intestinal microbiota from non-fermentative rods of the Cytophaga/Flexibacter/Flavobacterium group to fermentative bacteria of the Vibrio/Aeromonas group with the onset of first feeding on Artemia. The microbiology of the start-feeding turbot and halibut larvae thus differs from that of summer flounder, indicating that early larval microbiology can be site- and species-specific. Artemia used as feed for marine fish larvae can have high levels of vibrios (Austin and Allen, 1982; Verschuere et al., 1997, 1999; Olsen et al., 1999, 2000), and this was seen in the present study. The use of Artemia as a feed coincided with an increase in total and relative vibrio numbers in the water. The higher vibrio levels seen in the tanks during the Artemia stage may have been further encouraged by the presence of substrates favorable to vibrio proliferation, most notably, fish feces high in chitinous Artemia exoskeletons. A number of vibrios are known to be chitinolytic (West et al., 1986), and the vibrios are capable of survival and proliferation in fish feces (Olsson et al., 1998). The high vibrio levels seen in Artemia and stage 2 tank water were also observed in the intestines of stage 2 summer flounder. Muroga et al. (1987) found that in farmed red sea bream Pagrus major at a similar stage of development, about 45% of the intestinal bacteria were vibrios. Olsen et al. (1999, 2000) observed a positive correlation between the vibrio levels and types found in Artemia and those found in the halibut larvae consuming the

24

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Artemia. Summer flounder larvae also had vibrio types similar to those isolated from Artemia. In particular, V. alginolyticus, often the dominant strain in Artemia cultures, was found in 57% of the stage 2 summer flounder larvae. Blanch et al. (1997) showed that high levels of V. alginolyticus in Artemia cultures corresponded with the high levels in larvae feeding on the Artemia. This species disappeared from the fish when they were weaned to a formulated diet. Grisez et al. (1997) reported similar findings with sea bream Dicentrarchus labrax and sea bass Sparus aurata. The role of V. alginolyticus in fish health and disease remains unclear. It has been implicated as a pathogen (Austin et al., 1993; Sedano et al., 1996) and a probiotic (Austin et al., 1995; Grisez et al., 1997) and had no effect upon larval survival (Verdonck et al., 1997). Mortality rates were highest for stage 2 and stage 3 summer flounder larvae when Artemia was the predominant live feed. Although Artemia-associated vibrios were not implicated as the cause, previous studies have clearly indicated that vibrio pathogens can be transmitted via Artemia to larval fish (Muroga et al., 1990; Chair et al., 1994; Grisez et al., 1996; Sedano et al., 1996). The summer flounder gut undergoes important changes at metamorphosis, developing a true stomach, a longer and more complex intestine and greater digestive capability (Huang et al., 1998). Sugita et al. (1988) suggested that the development of an adult intestine was necessary for the establishment of a stable gut microbiota in the goldfish Carassius auratus. Digestive enzymes may exert a selective effect upon intestinal microbial populations (Grisez et al., 1997), and the metamorphosis may be the time when a truly indigenous gut microbiota becomes established in marine fish (Ringo and Birkbeck, 1999). Liston (1957) identified a group of luminescent vibrios as ‘‘gut group vibrios’’ because they were consistently isolated from marine flatfish intestines. These included V. fischerii and V. fluvialis, which in the present study were seen in 25% of the metamorphosing summer flounder but were detected in only 6% of stage 1 and stage 2 larvae. Increased levels of gut group vibrios may indicate the development of a gut microbiota more representative of adult fish. Artemia remained an important food item at this time, and V. alginolyticus was found in 33% of the metamorphosing fish. Stage 4 summer flounder have completed metamorphoses and are fully demersal. Gut group vibrios were detected in 38% of the stage 4 summer flounder, and an isolate characterized as V. fischerii was found in one fish at the highest numbers (2.3  106 CFU/ fish) enumerated for vibrio isolates at this stage. This was the first developmental stage where the fish vibrio index was higher than the water vibrio index. Bergh et al. (1994) and Muroga et al. (1987) inferred that higher vibrio levels detected in juvenile fish relative to culture water indicated the establishment of an indigenous gut microbiota. V. alginolyticus was not detected in any stage 4 fish, coincident with the cessation of Artemia as a feed and similar to the findings reported by Blanch et al. (1997) for turbot. A Bacillus spp. was detected in both the tank water and the fish intestine during stage 4. Gram-positive organisms are often found to be associated with marine fish but are typically not numerically significant (Liston, 1957; Cahill, 1990; Gatesoupe, 1999; Ringo and Birkbeck, 1999). Gatesoupe (1993) used Bacillus spp. spores for biocontrol and nutritional enhancement of rotifer cultures. Kennedy et al. (1998) isolated B. subtilis from the common snook, Centropomus undecimalis, and used this strain to suppress Vibrio spp.

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

25

in the rearing water. The Bacillus sp. found to be associated with the summer flounder was thus considered a probiotic candidate, and further work will examine this species as such.

5. Conclusion This study provides evidence that the microbiota associated with the fingerling production of summer flounder is similar in many respects to that observed for other marine fish species in culture. Trends consistent with those observed by other researchers were detected in microbial levels and composition of live feed organisms and fish developmental stages. A successional pattern was evident in the fish-rearing biotope, which can be summarized as a general increase in species diversity and levels of Vibrio spp. as the fish matured. This trend appeared to be influenced by the inputs of phytoplankton, rotifers and Artemia into the fish-rearing biotope, and the microbiota of the fish intestine also appeared to be affected by the live feed. Furthermore, larval rearing methods appeared to affect the tank microbial populations although the effects upon the microbial populations of the fish were not detected. Preliminary evidence was obtained, showing that the intestinal microbiota of summer flounder may become established as an autochthonous and stable population after metamorphosis, but further study is required for confirmation. The procedures used for microbial monitoring were effective and provide a basis for routine monitoring efforts designed to detect changes in microbial numbers and composition and allow informed decisions regarding microbial control measures. Furthermore, a large number of isolates from this facility were frozen and will be used in a future work to examine pathogenicity or probiotic potential, ultimately leading to the adoption of microbial control strategies that are not dependant upon chemotherapy.

Acknowledgements We thank Dr. Carroll Jones and Robert Gibson of the NH Veterinary Diagnostic Laboratory and the staff at GreatBay Aquafarms for the technical assistance. We also thank Ms. Beata Summer-Brason for the initial methods development and analyses. This study was supported by the UNH/UM Sea Grant Program (Project No. R/FMD-144), the Center for Marine Biology and the New Hampshire Industrial Research Center.

References Alsina, M., Blanch, A.R., 1994. A set of keys for biochemical identification of environmental Vibrio species. J. Appl. Bacteriol. 76, 79 – 85. Alsina, M., Martinez-Picado, J., Jofre, J., Blanch, A.R., 1994. A medium for presumptive identification of Vibrio anguillarum. Appl. Environ. Microbiol. 60, 1681 – 1683. Andrews, J.H., Harris, R.F., 1986. R- and K-selection in microbial ecology. Adv. Microb. Ecol. 9, 99 – 147. Austin, B., Allen, D.A., 1982. Microbiology of laboratory-hatched brine shrimp (Artemia). Aquaculture 26, 369 – 383.

26

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Austin, B., Al-Zahrani, A.M.J., 1988. The effect of antimicrobial compounds on the gastrointestinal microflora of rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 33, 1 – 14. Austin, B., Baudet, E., Stobie, M., 1992. Inhibition of bacterial fish pathogens by Tetraselmis suecica. J. Fish Dis. 15, 55 – 61. Austin, B., Stobie, M., Robertson, P.A.W., Glass, H.G., Stark, J.R., Mudarris, M., 1993. Vibrio alginolyticus—the cause of gill disease leading to progressive low-level mortalities among juvenile turbot, Scophthalmus maximus L. in a Scottish aquarium. J. Fish Dis. 16, 277 – 280. Austin, B., Stuckey, L.F., Robertson, P.A.W., Effendi, I., Griffith, D.R.W., 1995. A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J. Fish Dis. 18, 93 – 96. Baker, E.P., Alves, D., Bengston, D., 1998. Effects of rotifer and Artemia fatty-acid enrichment on survival, growth, and pigmentation of summer flounder Paralichthys dentatus larvae. J. World Aquacult. Soc. 29, 494 – 498. Bengston, D.A., 1999. Aquaculture of summer flounder (Paralichthys dentatus): status of knowledge, current research and future research priorities. Aquaculture 176, 39 – 49. Bengston, D.A., Nardi, G., 2000. Summer flounder culture. Encyclopedia of Aquaculture. Wiley, New York, pp. 907 – 913. Bergh, O., Naas, K.E., Harboe, T., 1994. Shift in the intestinal microflora of Atlantic halibut (Hippoglossus hippoglossus) larvae during first feeding. Can. J. Fish. Aquat. Sci. 51, 1899 – 1903. Blanch, A.R., Alsina, M., Simon, M., Jofre, J., 1997. Determination of bacteria associated with reared turbot (Scophthalmus maximus) larvae. J. Appl. Microbiol. 82, 729 – 734. Bolinches, J., Romalde, J.L., Toranzo, A.E., 1988. Evaluation of selective media for isolation and enumeration of vibrios from estuarine waters. J. Microbiol. Methods 8, 151 – 160. Cahill, M.M., 1990. Bacterial flora of fishes: a review. Microb. Ecol. 19, 21 – 41. Campbell, A.C., Buswell, J.A., 1983. The intestinal microflora of farmed Dover sole (Solea solea) at different stages of fish development. J. Appl. Bacteriol. 55, 215 – 223. Chair, M., Dehasque, M., Poucke, S.V., Nelis, H., Sorgeloos, P., De Leenheer, A.P., 1994. An oral challenge for turbot larvae with Vibrio anguillarum. Aquacult. Int. 2, 270 – 272. Douillet, P.A., Pickering, P.L., 1999. Seawater treatment for larval culture of the fish Sciaenops ocellatus Linnaeus (red drum). Aquaculture 170, 113 – 126. Gatesoupe, F.J., 1993. Bacillus sp. spores as food additive for the rotifer Brachionus plicatilis: improvement of their bacterial environment and their dietary value for larval turbot, Scophthalmus maximus L.. In: Kaushik, S.J., Luguet, P. (Eds.), Fish Nutrition in Practice. Les Colloques, vol. 61. Institut National de la Recherche Agronomique, Paris, France, pp. 561 – 568. Gatesoupe, F.J., 1994. Lactic acid bacteria increase the resistance of turbot larvae, Scophthalmus maximus, against pathogenic vibrio. Aquat. Living Resour. 7, 277 – 282. Gatesoupe, F.J., 1999. The use of probiotics in aquaculture. Aquaculture 180, 147 – 165. Gildberg, A., Mikkelsen, H., Sandaker, E., Ringo, E., 1997. Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua). Hydrobiologia 352, 279 – 285. Gram, L., Melchiorsen, J., Spanggaard, B., Huber, I., Nielsen, T.F., 1999. Inhibition of Vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish. Appl. Environ. Microbiol. 65, 969 – 973. Grisez, L., Chair, M., Sorgeloos, P., Ollevier, F., 1996. Mode of infection and spread of Vibrio anguillarum in turbot Scophthalmus maximus larvae after oral challenge through live feed. Dis. Aquat. Org. 26, 181 – 187. Grisez, L., Reyniers, J., Verdonck, L., Swings, J., Ollevier, F., 1997. Dominant intestinal microflora of sea bream and sea bass larvae, from two hatcheries, during larval development. Aquaculture 155, 387 – 399. Hameed, A.S., Balasubramanian, G., 2000. Antibiotic resistance in bacteria isolated from Artemia nauplii and efficacy of formaldehyde to control bacterial load. Aquaculture 183, 195 – 205. Hansen, G.H., Olafsen, J.A., 1989. Bacterial colonization of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus) eggs in marine aquaculture. Appl. Environ. Microbiol. 55, 1435 – 1446. Hansen, G.H., Olafsen, J.A., 1999. Bacterial interactions in early life stages of marine cold water fish. Microb. Ecol. 38, 1 – 26. Hansen, G.H., Strom, E., Olafsen, J.A., 1992. Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae. Appl. Environ. Microbiol. 58, 461 – 470.

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

27

Hoff, F.H., Snell, T.W., 1993. Plankton Culture Manual. Florida Aqua Farms, Florida 147 pp. Horne, M.T., Richards, R.H., Roberts, R.J., Smith, P.C., 1977. Peracute vibriosis in juvenile turbot Scophthalmus maximus. J. Fish Biol. 11, 355 – 361. Huang, L., Schreiber, A.M., Soffientino, B., Bengston, D.A., Specker, J.L., 1998. Metamorphosis of summer flounder (Paralichthys dentatus): thyroid status and the timing of gastric gland formation. J. Exp. Zool. 280, 413 – 420. Kapetenaki, M.J., Kerry, J., Hiney, M., O’Brien, C., Coyne, R., Smith, P., 1995. Emergence, in oxytetracyclinefree marine mesocosms, of microorganisms capable of colony formation on oxytetracycline-containing media. Aquaculture 134, 227 – 236. Kennedy, S.B., Tucker, J.W., Neidig, C.L., Vermeer, G.K., Cooper, V.R., Jarrell, J.L., Sennett, D.G., 1998. Bacterial management strategies for stock enhancement of warmwater marine fish: a case study with common snook (Centropomus undecimalis). Bull. Mar. Sci. 62, 572 – 588. Keskin, M., Keskin, M., Rosenthal, H., 1994. Pathways of bacterial contamination during egg incubation and larval rearing of turbot, Scophthalmus maximus. J. Appl. Ichthyol. 10, 1 – 9. Liston, J., 1957. The occurrence and distribution of bacterial types on flatfish. J. Gen. Microbiol. 16, 205 – 216. Maeda, M., 1994. Biocontrol of the larvae rearing biotope in aquaculture. Bull. Natl. Res. Inst. Aquacult. (Suppl. 1), 71 – 74. McLaughlin, J.C., 1995. Vibrio. In: Murray, P.R., Baron, E.J. (Eds.), Manual of Clinical Microbiology. ASM Press, Washington, DC, pp. 465 – 475. Munro, P.D., Barbour, A., Birkbeck, T.H., 1994. Comparison of the gut bacterial flora of start-feeding larval turbot reared under different conditions. J. Appl. Bacteriol. 77, 560 – 566. Munro, P.D., McLean, H.A., Barbour, A., Birkbeck, T.H., 1995a. Stimulation or inhibition of growth of the unicellular alga Pavlova lutheri by bacteria from larval turbot culture systems. J. Appl. Bacteriol. 79, 519 – 524. Munro, P.D., Barbour, A., Birkbeck, T.H., 1995b. Comparison of the growth and survival of larval turbot in the absence of culturable bacteria with those in the presence of Vibrio anguillarum, Vibrio alginolyticus, or a marine Aeromonas sp. Appl. Environ. Microbiol. 61, 4425 – 4428. Muroga, K., Higashi, M., Keitoku, H., 1987. The isolation of intestinal microflora of farmed red sea bream (Pagrus major) and black sea bream (Acanthopagrus schleheli) at larval and juvenile stages. Aquaculture 65, 79 – 88. Muroga, K., Yasunobu, H., Okada, N., Masumura, K., 1990. Bacterial enteritis of cultured flounder Paralichthys olivaceus larvae. Dis. Aquat. Org. 9, 121 – 125. Nicolas, J.L., Robic, E., Ansquer, D., 1989. Bacterial flora associated with a trophic chain consisting of microalgae, rotifers and turbot larvae: influence of bacteria on larval survival. Aquaculture 83, 237 – 248. Nogami, K., Maeda, M., 1992. Bacteria as biocontrol agents for rearing of the crab Portunus trituberculatus. Can. J. Fish. Aquat. Sci. 49, 2373 – 2376. Olsen, A.I., Attramadal, Y., Jensen, A., Olsen, Y., 1999. Influence of size and nutritional value of Artemia franciscana on growth and quality of halibut larvae (Hippoglossus hippoglossus) during the live feed period. Aquaculture 179, 475 – 487. Olsen, A.I., Olsen, Y., Attramadal, Y., Christie, K., Birkbeck, T.H., Skjermo, J., Vadstein, O., 2000. Effects of short-term feeding of microalgae on the bacterial flora associated with juvenile Artemia franciscana. Aquaculture 190, 11 – 25. Olsson, J.C., Westerdahl, A., Conway, P.L., Kjelleberg, S., 1992. Intestinal colonization potential of turbot (Scophthalmus maximus) and dab (Limanda limanda) associated bacteria with inhibitory effects against Vibrio anguillarum. Appl. Environ. Microbiol. 58, 551 – 556. Olsson, J.C., Joborn, A., Westerdahl, A., Blomberg, L., Kjelleberg, S., Conway, P.L., 1998. Survival, persistence and proliferation of Vibrio anguillarum in juvenile turbot, Scophthalmus maximus (L.), intestine and feces. J. Fish Dis. 21, 1 – 9. Pedersen, K., Dalsgaard, I., Larsen, J.L., 1997. Vibrio damsela associated with diseased fish in Denmark. Appl. Environ. Microbiol. 63, 3711 – 3715. Reitan, I.R., Rainuzzo, J.R., Gunvor, O., Olsen, Y., 1997. A review of the nutritional effects of algae in marine fish larvae. Aquaculture 155, 207 – 221. Rico-Mora, R., Voltolina, D., Villaescusa-Celaya, J.A., 1998. Biological control of Vibrio alginolyticus in Skeletonema costatum (Bacillariophyceae) cultures. Aquacult. Eng. 19, 1 – 6.

28

S.D. Eddy, S.H. Jones / Aquaculture 211 (2002) 9–28

Ringo, E., Birkbeck, T.H., 1999. Intestinal microflora of fish larvae and fry. Aquacult. Res. 30, 73 – 93. Ringo, E., Birkbeck, T.H., Munro, P.D., Vadstein, O., Hjelmeland, K., 1996. The effect of early exposure to Vibrio pelagius on the aerobic bacterial flora of turbot, Scophthalmus maximus (L.). J. Appl. Bacteriol. 81, 207 – 211. Riquelme, C., Hayashida, G., Vergara, N., Vasques, A., Morales, Y., Chavez, P., 1995. Bacteriology of the scallop Argopecten purpuratus (Lamarck, 1819) cultured in Chile. Aquaculture 138, 49 – 60. Riquelme, C., Araya, R., Vergara, N., Rojas, A., Guaita, M., Candia, M., 1997. Potential probiotic strains in the culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819). Aquaculture 154, 17 – 26. Sakai, M., Yoshida, T., Atsuta, S., Kobayashi, M., 1995. Enhancement of resistance to vibriosis in rainbow trout, Oncorhynhus mykiss (Walbaum), by oral administration of Clostridium butyricum bacterin. J. Fish Dis. 18, 187 – 190. Sedano, J., Zorrilla, I., Morinigo, M.C., Vidaurreta, A., Bordas, M.A., Borrego, J.J., 1996. Microbial origin of the abdominal swelling affecting farmed larvae of gilt-head sea bream, Sparus aurata L. Aquacult. Res. 27, 323 – 333. Skjermo, J., Vadstein, O., 1999. Techniques for microbial control in the intensive rearing of marine larvae. Aquaculture 177, 333 – 343. Skjermo, J., Salvesen, I., Oie, G., Olsen, Y., Vadstein, O., 1997. Microbially matured water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae. Aquacult. Int. 5, 13 – 28. Smith, P., Davey, S., 1993. Evidence for the competitive exclusion of Aeromonas salmonicida from fish with stress-inducible furunculosis by a fluorescent pseudomonad. J. Fish Dis. 16, 521 – 524. Sugita, H., Tsunohara, M., Ohkoshi, T., Deguchi, Y., 1988. The establishment of an intestinal microflora in developing goldfish (Carassius auratus) of culture ponds. Microb. Ecol. 15, 333 – 344. Verdonck, L., Grisez, L., Sweetman, E., Minkoff, G., Sorgeloos, P., Ollevier, F., Swings, J., 1997. Vibrios associated with routine productions of Brachionus plicatilis. Aquaculture 149, 203 – 214. Verschuere, L., Dhont, J., Sorgeloos, P., Verstraete, W., 1997. Monitoring biolog patterns and r/K-strategists in the intensive culture of Artemia juveniles. J. Appl. Microbiol. 83, 603 – 612. Verschuere, L., Rombaut, G., Huys, G., Dhont, J., Sorgeloos, P., Verstraete, W., 1999. Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains. Appl. Environ. Microbiol. 65, 2527 – 2533. Watanabe, W.O., Ellis, E.P., Ellis, S.C., 1998. Progress in controlled maturation and spawning of summer flounder Paralichthys dentatus broodstock. J. World Aquacult. Soc. 29, 393 – 404. West, P.A., Brayton, P.R., Bryant, T.N., Colwell, R.R., 1986. Numerical taxonomy of vibrios isolated from aquatic environments. Int. J. Syst. Bacteriol. 36, 531 – 543.