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Analysis of bacterial communities in Nannochloropsis sp. cultures used for larval fish production Gentoku NAKASE AND Mitsuru EGUCHI* Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan ABSTRACT: Phytoplankton used in fish hatcheries is mass-cultured in the open air and usually contains large numbers of bacteria. In commercial fish production, the phytoplankton cultures are usually added into the larval rearing tanks; however, the numbers and types of bacteria introduced into the rearing tanks simultaneously are unknown. In this study, the bacterial community structures in Nannochloropsis sp. cultures were analyzed by using fluorescence in situ hybridization (FISH). A direct viable count (DVC)-FISH analysis was also performed as DVC is useful for the detection of actively growing cells. Total numbers of bacteria in Nannochloropsis sp. cultures ranged from 7.72 ¥ 105-2.39 ¥ 106 cells/mL. High proportions of the total bacteria (31.6–53.6%) in the Nannochloropsis sp. cultures showed growth potential. DVC-FISH analysis revealed that a-proteobacteria and the Cytophaga–Flavobacterium cluster were abundant in the bacterial community of actively growing cells. Thus, the high growth potentials of the distinct bacterial communities in Nannochloropsis sp. culture must influence the bacterial communities in larval rearing tanks. KEY WORDS: bacterial community structure, biocontrol, direct viable count (DVC), fluorescence in situ hybridization (FISH), larval rearing, Nannochloropsis, rearing water.

INTRODUCTION In fish hatcheries, the addition of phytoplankton to the rearing tanks of fish larvae enhances larval survival and growth.1–3 Phytoplankton is masscultured in the open air; therefore, it is impossible to avoid bacterial contamination and to keep the culture axenic. Thus, phytoplankton cultures usually contain large numbers of bacteria. Nicolas et al.4 estimated total bacterial numbers in several phytoplankton cultures, such as Pavlova lutheri, Isochrysis galban (haptophytes), Skeletonema costatum, Chaetoceros calcitrans, and Chaetoceros gracilis (diatoms), and showed that densities varied from 1.3 ¥ 105 to 5.3 ¥ 108 cells/ mL. Thus, the addition of a phytoplankton culture also means the mass introduction of bacteria into the larval rearing system. However, there is little qualitative data on bacteria in phytoplankton cultures used for fish larvae productions.

*Corresponding author: Tel: 81-742-43-6354. Fax: 81-742-43-6354. Email: [email protected] Received 24 November 2006. Accepted 5 January 2007.

doi:10.1111/j.1444-2906.2007.01366.x

The bacterial community structure is a key factor affecting the survival of fish larvae.5–7 It is inevitable that phytoplankton addition will affect the bacterial community in the rearing water. Therefore, addition of phytoplankton can control bacterial communities, as reported previously.2,4,8 Control of the bacterial community during addition of phytoplankton should improve larval survival; however, this is not yet proven.2 Nicolas et al.4 also analyzed the communities of culturable bacteria in the phytoplankton cultures. However, studies of this type are few and for Nannochloropsis cultures, a phytoplankton often used in fish hatcheries, there are no data on bacterial community structures. Thus, the abundance and phylogenetic groups of bacteria introduced into the rearing tanks by Nannochloropsis culture are unknown. In this study, bacterial community structures in Nannochloropsis sp. cultures were analyzed using fluorescence in situ hybridization (FISH). In addition to the standard FISH analysis, we also performed FISH combined with direct viable count (DVC)9 which is useful for the detection of actively growing cells.10 © 2007 Japanese Society of Fisheries Science

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Table 1 Cell densities and incubation temperatures of the Nannochloropsis sp. cultures from Malaysia Cell number (cells/mL) Culture 1 Culture 2 Culture 3

Water temperature (°C)

7

1.34 ¥ 10 7.80 ¥ 106 9.10 ¥ 106

32.1 29.7 29.9

Experiments were performed twice: culture 1 was from the first experiment and cultures 2 and 3 were from the second experiment.

MATERIALS AND METHODS Nannochloropsis culture in Malaysia Nannochloropsis sp. was cultured at the Borneo Marine Research Institute, University Malaysia Sabah, Malaysia, for use in fish larvae production. Nannochloropsis sp. culture (100 L) and sandfiltered natural seawater (100 L) were mixed in a 500-L white resin tank. The experiments were performed twice. One culture (culture 1) was analyzed in the first experiment and two cultures (cultures 2 and 3) were analyzed in the second experiment. The algal cultures were aerated and cultured in the open air. The Nannochloropsis sp. cultures were enriched by addition of agricultural fertilizer (ammonium sulfate, urea, and superphosphate of lime) at the beginning of the experiments. Samples were taken from the three tanks when the Nannochloropsis sp. cultures were in the logarithmic growth phases. Cell counts of the Nannochloropsis sp. cultures were estimated using a Thoma type blood-counting chamber (Hirschmann, Eberstadt, Germany). Temperatures of Nannochloropsis sp. cultures were measured using a mercury thermometer (Table 1). Nannochloropsis sp. culture in Japan Nannochloropsis sp. for commercial fish larvae production was cultured at the Kinki University Fish Nursery Center, Kinki University, Wakayama, Japan. Nannochloropsis sp. was cultured in 2000-L transparent polycarbonate tanks in the open air. The culture in the logarithmic growth phase was sampled and cell density was estimated by using a Thoma blood-counting chamber (Table 2). Water temperatures were recorded at the same time. Sampling for microbial analysis Nannochloropsis sp. culture samples were filtered through a 3-mm pore size polycarbonate filter to © 2007 Japanese Society of Fisheries Science

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Table 2 Cell numbers of Nannochloropsis sp. and bacteria in the Nannochloropsis sp. culture in Shirahama, Wakayama, Japan Incubation temperature (°C) Nannochloropsis cell number (cells/mL) Total bacterial number (cells/mL) DVC+ cell number (cells/mL) DVC+/total (%)

20.0 7.72 ¥ 106 2.39 ¥ 106 7.56 ¥ 105 31.6

DVC+, DVC-positive; DVC+/total, proportion of DVC+ cells to total bacteria.

remove Nannochloropsis sp. cells. Each filtrate was collected and divided into two subsamples. One subsample was fixed with 20% paraformaldehyde– PBS (pH 7.2) (final concentration in samples 2%) immediately after sampling. The other subsample was used for direct viable count (DVC) analysis. The fixed samples were used for enumeration of total bacteria and analysis of bacterial community structures by FISH after fixation for 24 h.

Total bacterial count Total bacterial numbers in the samples were enumerated by direct-count method using the fluorochrome 4′,6-diamidino-2-phenylindole (DAPI, final concentration 0.5 mg/mL).11 Bacterial cells in the samples were filtered onto 0.2-mm pore size polycarbonate filters and enumerated using an epifluorescence microscope (BX-51, Olympus, Tokyo, Japan) under UV excitation (Fig. 1a).

Fluorescence in situ hybridization (FISH) Bacterial community structures were analyzed by FISH with rRNA-targeted oligonucleotide probes labeled with fluorochrome (Cy3). The fixed cells were filtered onto 0.2-mm pore size filters and FISH was performed according to the method of Glöckner et al.12 The probes used in this study were ALF1b for a-proteobacteria,13 GAM42a for g-proteobacteria,13 and CF319a for the CytophagaFlavobacterium cluster.14 Hybridization was performed on the filters for 3 h at 46°C and then the filters were washed for 15 min at 48°C. After washing, bacterial DNA in the samples was stained with DAPI. The bacterial cells, which had both hybridization signals and DAPI fluorescence, were counted as hybridized cells. Relative abundance of each specific bacterial group was expressed as the ratio of hybridized cells to total DAPI-stained cells. DAPI-stained cells that did not hybridize with the probes were classified as unknown.

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exin (10 mg/mL), and ciprofloxacin (0.5 mg/mL) were added to the samples as inhibitors of cell division. As we did not want to change the conditions of naturally occurring organic substances in the Nannochloropsis sp. cultures, we did not add any other organic nutrients such as yeast extract into the DVC samples. The DVC samples were then incubated at in situ temperature in the dark for 16 h and fixed with 20% paraformaldehyde–PBS (final concentration 2%). Then, all bacterial cells in the DVC samples were stained with DAPI and counted. Total bacterial numbers in the DVC samples were compared with those in the samples fixed immediately after sampling to confirm that no cell division occurred after addition of antibiotics. Cell sizes in the DVC samples were determined using image-processing software (Win ROOF, Mitani Co., Tokyo, Japan) from photomicrographs. Cell sizes in the samples fixed immediately after sampling (before incubation) were also determined. DVC cells that were more than twice as large as the average sizes of cells before incubation were considered as DVC-positive (DVC+)16 (Fig. 1b).

DVC-FISH

(c)

Bacterial community structures of actively growing cells in Nannochloropsis sp. cultures were analyzed by applying the FISH method to DVC samples. Cells more than double in size, which had both Cy3 and DAPI fluorescence, were considered to be probe-combined and actively growing cells (Fig. 1b,c). Cells stained with DAPI and elongated but not combined with any probes were classified as unknown.

RESULTS

Fig. 1 Epifluorescence photomicrographs of bacterial cells in Nannochloropsis sp. cultures. (a) DAPI-stained cells before DVC incubation, (b) DAPI-stained cells after 16-h DVC incubation, and (c) probe-reacted (ALF1b) cells after 16-h DVC incubation. Bars, 10 mm.

Direct viable count An improved DVC method10,15 was used to detect active bacterial cells in the Nannochloropsis sp. cultures. Five types of antibiotics: nalidixic acid (final concentration 20 mg/mL), piromidic acid (10 mg/mL), pipemidic acid (10 mg/mL), cephal-

In the culture experiment conducted in Malaysia, total numbers of bacteria and DVC-positive cells in Nannochloropsis sp. cultures ranged 7.72 ¥ 105– 1.70 ¥ 106 and 4.14 ¥ 105–8.60 ¥ 105 cells/mL, respectively (Table 3). High proportions of DVC positive cells relative to total bacteria (48.1–53.6%) indicated high growth potential in Nannochloropsis sp. cultures in Malaysia (Table 3). Bacterial community structures in the Nannochloropsis sp. cultures were analyzed by FISH. The ratios of the cells detected with the probes ALF1b, GAM42a, and CF319a, to total bacteria ranged 4.5–26.9% (average 19.6%), 5.5–7.7% (average 6.4%), and 3.6–9.9% (average 6.5%), respectively. The unknown group, which did not © 2007 Japanese Society of Fisheries Science

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Table 3 Numbers of total and DVC-positive bacterial cells in the Nannochloropsis sp. cultures from Malaysia

Culture 1 Culture 2 Culture 3

Total (cells/mL)

DVC+ (cells/mL)

DVC+/total (%) (cells/mL)

1.70 ¥ 106 1.41 ¥ 106 7.72 ¥ 105

8.60 ¥ 105 6.78 ¥ 105 4.14 ¥ 105

50.6 48.1 53.6

Total, total bacterial cells; DVC+, DVC-positive; DVC+/total, proportion of DVC+ cells to total bacteria. Experiments were performed twice: culture 1 was from the first experiment and cultures 2 and 3 were from the second experiment.

react with any probes, accounted for 57.8–86.0% of total bacteria (average 67.5%) (Fig. 2a). DVC-FISH revealed that the relative abundance of actively growing cells detected by ALF1b, GAM42a, and CF319a to total DVC positive cells ranged 55.4–68.1% (average 60.6%), 7.8–10.8% (average 9.4%), and 5.3–5.9% (average 5.6%), respectively (Fig. 2b). More than 70% of the actively growing cells in the Nannochloropsis sp. cultures used for fish larvae production in Malaysia could be detected with the three probes used in this study. Among the three types of bacteria, a-proteobacteria was predominant in the actively growing bacterial communities. Bacterial community structure in Nannochloropsis sp. cultures from Shirahama, Wakayama, Japan was also analyzed. FISH analysis showed that the cells detected with the probes ALF1b, GAM42a, and CF319a involved 12.4, 2.4, and 8.1% of total bacteria in the Nannochloropsis sp. culture, respectively (Fig. 3). The ‘unknown’ fraction, which was undetectable with any probe used in this study, was abundant, involving 77.1% of total bacteria (Fig. 3). DVC-FISH analysis revealed that a-proteobacteria (29.1%) and the Cytophaga– Flavobacterium cluster (38.7%) were abundant and the ‘unknown’ group was 25.7% in the bacterial community of actively growing cells (Fig. 3). In the Nannochloropsis sp. culture from Shirahama, the counts of total bacteria and actively growing cells were 2.39 ¥ 106 and 7.56 ¥ 105 cells/mL, respectively. The percent of actively growing cells to total bacteria was 31.6% (Table 2).

DISCUSSION High growth potential The total numbers of bacteria in the Nannochloropsis sp. cultures examined in this study ranged 7.72 ¥ 105-2.39 ¥ 106 cells/mL (Tables 2 and 3); these values were generally in the same range of © 2007 Japanese Society of Fisheries Science

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bacterial counts in coastal sea waters (from 2.78 ¥ 105 to 5.75 ¥ 106 cells/mL).15,17,18 The proportion of DVC positive cells to total bacteria in the Nannochloropsis sp. cultures averaged 46.0%. The equivalent values in natural coastal seawater samples ranged 6.9–44.4% (average 25.4%).15 Bacterial cells in Nannochloropsis sp. cultures were more active than in natural coastal sea water. Distinct bacterial communities In the bacterial communities with high growth potential in the Nannochloropsis sp. cultures, aproteobacteria and the Cytophaga–Flavobacterium cluster were predominant (Figs 2b and 3). These results, the predominance of a-proteobacteria and the Cytophaga–Flavobacterium cluster in DVC samples, suggest a close relationship between Nannochloropsis sp. and bacteria in culture. In diatom and dinoflagellate cultures, Nicolas et al.,4 Grossart et al.,19 and Jasti et al.20 also showed the predominance of a-proteobacteria mainly composed of Roseobacter and the Cytophaga–Flavobacterium cluster, and discussed the close relationship between bacteria and phytoplankton. Thus, even in Nannochloropsis sp. cultures, in which aproteobacteria dominated in the communities of actively growing bacteria, a type of Roseobacter sp. may also be present. Phytoplankton produce two types of substrates: one is available for bacteria21,22 and the other inhibits bacterial growth.23–25 Roseobacter sp. consumes dimethylsulfoniopropionate (DMSP), which is produced and excreted by some phytoplankton species.26 Polysaccharides are produced by diatoms27 and the polysaccharides are degraded by members of Cytophagacaea.28 In the present study, excreta of Nannochloropsis sp. may also have activated the growth of a-proteobacteria and/or the Cytophaga–Flavobacterium cluster selectively. Application of DVC without addition of organic nutrients DVC samples are usually added with organic nutrients to promote cell division. In this study, any organic nutrients such as yeast extract were not added to DVC samples, as we did not want to change the conditions of naturally occurring organic substances in the Nannochloropsis sp. cultures. Addition of organic nutrients causes the change of bacterial community structures in seawater samples.29 In this study, a high proportion (average 46.0%) of DVC-positive cells (Tables 2 and 3) were detected without addition of organic

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(a)

Culture 1

Culture 2

Culture 3

Culture 1

Culture 2

Culture 3

(b)

Fig. 2 Bacterial community structures in Nannochloropsis sp. cultures from Malaysia. (a) FISH analysis without DVC, (b) FISH analysis with DVC. Experiments were performed twice, culture 1 was from the first experiment and cultures 2 and 3 were from the second experiment; ‘unknown’ indicates bacterial cells that did not combine with any oligonucleotide probe used in this study.

α- proteobacteria

Cytophaga-Flavobacterium cluster

γ- proteobacteria

unknown

nutrients. DVC without addition of organic nutrients can be useful to detect active growing cells from the natural environment without bias by addition of organic nutrients. Influences of addition of Nannochloropsis sp. on bacterial community in rearing water In the seawater rearing of larval fish, microalgal cultures are often added to the rearing waters for increasing the survival rate of larvae. Then, active bacterial populations are also introduced into the rearing waters by the Nannochloropsis sp. addition. The introduced bacteria, which have been concomitant with the Nannochloropsis sp., must affect the bacterial communities in larval rearing water, especially during the early rearing operation with a low water exchange rate. The influence

of bacterial communities in rearing tanks on the survival of fish larvae has been discussed previously.5–7 For successful larval rearing, the stable balance of a-proteobacteria and the Cytophaga– Flavobacterium cluster seems to be required (Nakase et al., in press, 2007). The addition of the Nannochloropsis sp. cultures dominated by actively growing a-proteobacteria and the Cytophaga– Flavobacterium cluster could be beneficial in keeping the stable balance of a-proteobacteria and the Cytophaga–Flavobacterium cluster in rearing systems. It is believed that the addition of phytoplankton cultures into larval fish rearing tanks has some advantages, such as the supply of beneficial nutrients for the growth of rotifers,30,31 enhancement of feeding with increased turbidity,32,33 and stabilization of water qualities.2 The results of the present study show an additional advantage that the © 2007 Japanese Society of Fisheries Science

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(a)

(b)

α- proteobacteria γ- proteobacteria

Cytophaga-Flavobacterium cluster unknown Fig. 3 Bacterial community structures in Nannochloropsis sp. culture from Shirahama, Wakayama, Japan. Bacterial community structures analyzed by (a) FISH and (b) DVC-FISH; ‘unknown’ indicates bacterial cells that did not combine with any oligonucleotide probe used in this study.

bacterial communities in the Nannochloropsis sp. cultures had active growth potential and were dominated by a-proteobacteria and/or the Cytophaga–Flavobacterium cluster. The addition of Nannochloropsis sp. culture dominated by actively growing a-proteobacteria and the Cytophaga– Flavobacterium can be a useful tool for biocontrol of the bacterial communities in fish larval rearing systems.

ACKNOWLEDGMENTS We thank Professor S. Miyashita and Dr. Y. Nakagawa of the Fisheries Laboratory, Kinki University for providing the Nannochloropsis culture used in this study. We also thank Professor Senoo and all staff of Borneo Marine Research Institute, University Malaysia Sabah for their kind help and advice. This study was partly supported by Research Fellowships of the Japanese Society for the Promotion of Science (JSPS) for Young Scientists (No. 1653311), a Grant-in Aid (19580222) from JSPS, and 21st century COE program (Center of Aquaculture Science and Technology for Bluefin Tuna and Other Cultivated Fish) of the Ministry of Education, Culture, Sports, Science and Technology, Japan. © 2007 Japanese Society of Fisheries Science

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