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Applied and Environmental Microbiology, August 2005, p. 4307-4317, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4307-4317.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effects of Bacteria on Artemia franciscana Cultured in Different Gnotobiotic Environments

Antonio Marques,^1* Thi Dinh,¹ Christos Ioakeimidis,¹ Geert Huys,² Jean Swings,^2,3 Willy Verstraete,⁴ Jean Dhont,¹ Patrick Sorgeloos,¹ and Peter Bossier¹

Laboratory of Aquaculture and Artemia Reference Center, Faculty of Bioscience Engineering, Ghent University, Rozier 44, 9000 Ghent, Belgium,¹ Laboratory of Microbiology,² BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium,³ Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium⁴

Received 16 December 2004/ Accepted 10 March 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The use of probiotics is receiving considerable attention as an alternative approach to control microbiota in aquaculture farms, especially in hatching facilities. However, application with consistent results is hampered by insufficient information on their modes of action. To investigate whether dead bacteria (allowing investigation of their nutritional effect) or live bacteria (allowing evaluation of their probiotic effect) have any beneficial effect towards Artemia franciscana and, subsequently, if live bacteria have probiotic effects beyond the effects observed with dead bacteria, a model system was employed using gnotobiotic Artemia as a test organism. Nauplii were cultured in the presence of 10 bacterial strains combined with four different major axenic live feeds (two strains of Saccharomyces cerevisiae and two strains of Dunaliella tertiolecta) differing in their nutritional values. In combination with poor- and medium-quality live feeds, dead bacteria exerted a strong effect on Artemia survival but a rather weak or no effect on individual length and constituted a maximum of only 5.9% of the total ash-free dry weight supplied. These effects were reduced or even disappeared when medium- to good-quality major feed sources were used, possibly due to improvements in the health status of Artemia. Some probiotic bacteria, such as GR 8 (Cytophaga spp.), improved (not always significantly) the performance of nauplii beyond the effect observed with dead bacteria, independently of the feed supplied. The present approach can be an excellent system to study the exact mode of action of bacteria, especially if combined with challenge tests or other types of analysis (e.g., transcriptome and proteonomic analysis).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mass production of juvenile stages of aquatic organisms and their live preys for aquaculture obliges the use of intensive culture systems, which often leads to high mortalities caused mostly by pathogenic or opportunistic bacteria (5). The addition of high amounts of feed to these systems constitutes an excellent medium for the proliferation of heterotrophic bacteria, including opportunistic pathogens (49).

Until recently, one of the most frequent procedures used to avoid the establishment of undesirable bacteria in a target organism was the administration of antibiotics in the water (7). However, this practice promotes the selection and dissemination of antibiotic-resistant bacteria in the target organism and throughout the environment (19). For this reason, there is an urgent need to control the microbiota in hatching facilities by using alternative approaches.

Among other options (such as immunostimulation), the use of probiotics is receiving considerable attention (3, 15, 18, 22, 34, 52, 53). The application of a single probiotic strain or a combination of strains either in the rearing water or in live feed aims at a permanent or transient beneficial colonization of the host. However, none of those studies have provided sufficient evidence on the mode of action of the so-called "probionts" to allow for their application with consistent beneficial effects to the larvae. These effects could be partly, or entirely, due to a general improvement of the larval condition, for instance, as a consequence of the addition of essential nutrients by probionts. Moreover, although antimicrobial effects have already been shown in vitro (41), a clear demonstration of the causal link between beneficial effects of the probiont and in vivo suppression of a pathogen is rarely provided. According to Verschuere et al. (52), the two putative features of a probiont (i.e., nutritional and disease control) should be examined separately, although it is conceivable that a combination of a nutritional effect and disease control yields the best probiotic effect.

In order to study the effects of microorganisms more accurately, a model system was employed using the brine shrimp, Artemia franciscana, as a test organism. Artemia is one of the most important live feeds for commercial production of fish and shellfish larvae (45). This organism can be fed on a wide variety of feeds since it is a continuous nonselective and particle filter feeder (45). The most commonly used feed sources include live microalgae, such as Dunaliella tertiolecta (11, 31), and baker's yeast (11), since they provide the basic nutrients for development of nauplii. Artemia is an excellent model organism to study the modes of action of probiotic and pathogenic bacteria, as it can easily be cultured under gnotobiotic conditions (29, 30) and can be used as a vector for transferring probiotics to larvae of target species.

The aim of the present study was not to select the probiotic strains to improve the mass rearing of the shrimp but to investigate whether dead or live bacteria have any beneficial effect towards Artemia and if live bacteria can have additional effects beyond those observed with dead bacteria. For that purpose, 10 different bacterial strains were tested dead or alive in a model system of gnotobiotic Artemia culture. These bacteria were combined with four different axenic live feeds with different nutritional values for Artemia: poor-quality feed (wild-type [WT] yeast), medium-quality feed (mnn9 yeast and the microalga DT CCAP 19/27), and good-quality feed (microalga DT CCAP 19/6B) (29, 30).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Axenic cultures of yeast and algae.
Two strains of axenic baker's yeast (Saccharomyces cerevisiae) were used as feed for Artemia: the WT strain (BY4741 [genotype, Mata his31 leu20 met150 ura30]) and its mnn9 isogenic mutant (BY4741 [genotype, Mata his31 leu20 met150 ura30 YPL050c::kanMX4]), which has a null mutation resulting in a lower concentration of mannose linked to mannoproteins and higher concentrations of chitin and glucans in the cell wall (2, 27, 29). Both strains were provided by the European Saccharomyces cerevisiae Archive for Functional Analysis, University of Frankfurt, Frankfurt, Germany. Yeast cultures were grown in sterile Erlenmeyer flasks with a cotton cap placed on a shaker in the dark (30°C, 150 rpm). Both strains were cultured in a complete yeast extract-peptone-dextrose medium containing yeast extract (composed of 1% [1% wt/vol] yeast extract, 1% [wt/vol] bacteriological-grade peptone, and 2% [wt/vol] D-glucose; Sigma), bacteriological-grade peptone (1%, wt/vol; Sigma), and D-glucose (2%, wt/vol; Sigma). This medium was prepared in natural seawater (35 g/liter) previously filtered (0.22-µm filter) and sterilized by autoclaving at 120°C for 20 min. The growth curve of each yeast strain was established by regularly measuring the absorbance at 600 nm with a spectrophotometer. Both strains were harvested by centrifugation (±800 x g for 10 min) in the stationary growth phase (after 3 days of culture, starting from a single colony). Cells were resuspended twice in sterile Falcon tubes (FT) (TRP) (-irradiated) with 20 ml of filtered and autoclaved seawater (FASW) (0.22 µm). All handlings were performed in a laminar flow hood to maintain sterility.

Axenic cultures of two strains of the microalga Dunaliella tertiolecta (strains DT CCAP 19/6B and DT CCAP 19/27) were obtained from the Culture Collection of Algae and Protozoa Department (CCAP), Dunstaffnage Marine Laboratory, Scotland. According to Marques et al. (30), both D. tertiolecta strains are identical in terms of the DNA sequence using the nuclear rRNA gene internal transcribed spacer region 2, indicating that they are phylogenetically very close. Each strain was grown in sterile autoclaved 500-ml bottles (10% inocula) with 0.22-µm-filtered aeration at 19°C and continuous light (±41 µEm^–2), using a standard Walne medium (54) and FASW. The growth curve of each algal strain was obtained by measuring the cell density of each culture daily using a Bürker hemocytometer, complemented with daily measurements of the optical density using a spectrophotometer ( = 600 nm). Algal strains were harvested in the exponential growth phase by centrifuging the culture (±800 x g for 5 min) in the middle of the exponential growth curve. Cultures were resuspended in 20 ml FASW in sterile Falcon tubes.

Yeast and microalga densities were determined by measuring the cell concentration twice using a Bürker hemocytometer. The suspensions were stored at 4°C and used to feed Artemia until the end of each experiment.

Bacterial strains and growth conditions.
A selection of 10 bacterial strains was examined: strains LVS 2, LVS 3, and LVS 8, for their positive effect towards Artemia (50, 51); strains GR 8, GR 10, GR 11, and GR 12, for their positive effect towards Brachionus plicatilis (42); a representative of cluster A, for its positive effect in turbot larviculture (22); Vibrio proteolyticus strain CW8T2, for its negative effect towards Artemia (50, 51); and Vibrio campbellii strain LMG21363, for its negative effect towards Artemia and shrimp (16, 46). Pure cultures of the 10 bacterial strains, stored at –80°C, were grown overnight at 28°C on marine agar (MA) containing Difco marine broth 2216 (37.4 g/liter; BD Biosciences) and bacteriological-grade agar (20 g/liter; ICN). For each bacterial strain, a single colony was selected from the plate and incubated overnight at 28°C in Difco marine broth 2216 on a shaker (150 rpm). Bacteria were harvested by centrifugation (15 min, ±2,200 x g), the supernatant was discarded, and the pellet was resuspended in 20 ml FASW. The densities of the new bacterial suspensions were determined by measuring their optical density at 550 nm with a spectrophotometer, assuming that an optical density of 1.000 corresponds to 1.2 x 10⁹ cells/ml, according to the McFarland standard (BioMerieux, Marcy L'Etoile, France).

Characterization of the bacterial strains.
Five beneficial bacterial strains were previously characterized (Table 1) based on their 16S rRNA gene sequencing (LVS 3, GR 8, GR 10, GR 11, and GR 12) by Makridis et al. (28). In the present work, the remaining strains, LVS 2, LVS 8, and a representative of cluster A, were characterized by 16S rRNA gene sequencing. For this purpose, genomic DNA was prepared according to the protocol of Pitcher et al. (37). 16S rRNA gene amplification, purification, and sequencing were performed as previously described (48), with the following modifications. 16S rRNA gene amplicons were purified by using a NucleoFast 96 PCR Clean-up kit (Macherey-Nagel). Sequencing reactions were performed by using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and purified using a Montage SEQ₉₆ Sequencing Reaction Clean-up kit (Millipore). Electrophoresis of sequence reaction products was performed by using an ABI Prism 3100 genetic analyzer (Applied Biosystems). Sequence assembly was performed using the program AutoAssembler (Applied Biosystems). Online similarity searches were performed with the BLAST (Basic Local Alignment Search Tool) family of programs in GenBank.

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TABLE 1. Identification of all bacterial strains tested and their average AFDWa

Killing of the bacteria.
Dead bacteria offered to Artemia were previously killed by using either 10-kGy -irradiation (provided by a 15-MeV 20-kW linear electron accelerator), 40% norvanol D (composed of 90% [vol/vol] ethanol, 2.9% [vol/vol] ether, and 9% [mass/vol] H₂O; Merck Eurolab) (incubated for 30 min at 28°C, centrifuged twice to remove norvanol residues, and replaced with FASW), or high temperature (autoclaving at 120°C for 20 min). For the two pathogenic strains (V. campbellii and V. proteolyticus), only autoclaving was used to obtain dead bacteria. In order to check if the bacteria were effectively killed by the three methods, all strains were plated after being exposed to each method by transferring 100 µl of the culture medium to MA (n = 3). Absence of bacterial growth was monitored after incubating plates for 5 days at 28°C. Autoclaving and -irradiation treatments were 100% effective, since no bacterial growth was observed on the MA after 5 days of incubation. As to the norvanol treatment, it was not effective in the gram-positive strain LVS 2. For this reason, strain LVS 2 treated with norvanol was not used in the four experiments. Dead and live bacterial suspensions were stored at 4°C until the end of each experiment (being supplied only once to Artemia at the beginning of the experiment).

Ash-free dry weight content.
To determine the ash-free dry weight (AFDW) content of live and dead bacteria (killed by either autoclaving, norvanol treatment, or -irradiation) and of the four feeds added to Artemia (yeast and algae), 50 ml of each culture sample was filtered on predried nitrocellulose filters (0.22-µm pore size; n = 2). Filters were subsequently dried at 60°C for 48 h and weighed. Afterwards, they were combusted at 600°C for 6 h to determine the ash content. The AFDW was calculated as the difference between the dry weight and the ash weight. The dry weight and AFDW of the control (filter only with the culture medium; n = 2) were subtracted from all samples.

Artemia gnotobiotic culture.
Experiments were performed with Artemia franciscana cysts, originating from Great Salt Lake, Utah (EG type; INVE Aquaculture NV, Belgium). Bacterium-free cysts and nauplii were obtained via decapsulation according to the procedure described previously by Sorgeloos et al. (45). During decapsulation, 0.22-µm-filtered aeration was provided. All manipulations were carried out under a laminar flow hood, and all necessary tools were previously autoclaved at 120°C for 20 min. Decapsulated cysts were washed carefully with FASW over a 50-µm-pore-size sterile net and transferred to a sterile 50-ml screw-cap Falcon tube containing 30 ml of FASW. The tube was capped and placed on a rotator at 4 cycles per min and exposed to constant incandescent light (±41 µEm^–2) at 28°C for 18 to 20 h. After this period, the hatching tube was taken to the laminar flow hood, and 20 hatched nauplii (Instar II) were transferred to new sterile 50-ml Falcon tubes containing 30 ml of FASW, together with the amount of feed scheduled for day 1. Each treatment consisted of four Falcon tubes (replicates). After feeding, the bacterial suspension (dead or live) was added only at day 1 at a density of approximately 5 x 10⁶ cells/ml in treatments where bacteria were used. All Falcon tubes were put back on the rotator and were transferred to the laminar flow hood just once per day for feeding. The daily feeding schedule was adapted from methods described previously by Coutteau et al. (11) and Marques et al. (30), who optimized the feeding schedule of Artemia using baker's yeast and D. tertiolecta as feed. The feeding schedule is intended to provide ad libitum ratios while avoiding excessive overfeeding in order not to affect the water quality in the test tubes. As control treatments, Artemia cultures were fed only yeast or algae without the addition of bacteria.

Methods used to verify axenity.
Axenity of feed, decapsulated cysts, and Artemia cultures were checked at the end of each experiment using a combination of plating and live counting according to the procedures of Marques et al. (29, 30). Absence of bacteria was monitored by transferring 100 µl of culture medium to petri plates with marine agar 2216 (n = 2). Plates were incubated for 5 days at 28°C. As for live counting, each sample was stained with tetrazolium salt MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] (0.5%, wt/vol; Sigma) in a sterile recipient (1 part MTT to 9 parts sample) and incubated at 30°C for 30 min. Under a light microscope (x1,000 magnification), live bacterial detection was performed. The MTT blue stains all viable/living cells remaining in a culture (44), making the detection of bacterial contaminations in a culture medium easier. A combination of plating and live counting was also used to detect contaminations in treatments where live bacteria were supplied to Artemia. The characteristics of the live bacteria present in these treatments were compared to the known characteristics of the bacterial strains used in order to verify the similarity between them. Whenever a culture tube was found to be contaminated, data were rejected and the treatment was repeated, as well as its axenic control.

Experimental design.
In experiment 1, 10 bacterial strains were tested dead or live on Artemia fed with the poor-quality WT yeast. In experiments 2 and 3, the medium-quality feeds (mnn9 yeast and the microalga DT CCAP 19/27, respectively) were used as feed for Artemia and inoculated with the same 10 bacterial strains added dead or live. Finally, in experiment 4, the same 10 bacterial strains were added dead or live to Artemia fed with the good-quality microalga DT CCAP 19/6B. In each experiment, performance of Artemia in the control treatments was compared to results previously obtained by Marques et al. (29, 30) to evaluate reproducibility. If significant differences were detected, data were not considered for further analysis and the experiment was repeated.

Survival and growth of Artemia.
At the end of each experiment (day 6 after hatching), the number of swimming larvae was determined and the survival percentage was calculated. Living larvae were fixed with Lugol's solution to measure their individual length (IL) using a dissecting microscope equipped with a drawing mirror, a digital plan measure, and the software Artemia 1.0 (courtesy of Marnix Van Damme). As a criterion that combines both the effects of survival and IL, the total biomass production (TBP or total length) was determined according to the following equation: TBP (millimeters per FT) = number of survivors x mean IL.

Statistics.
Values of larval survival (percentage) were arc sine transformed, while values of IL, TBP, and AFDW were logarithmic or square root transformed to satisfy normality and homocedasticity requirements. Differences between AFDW of dead and live bacteria and differences between survival, IL, and TBP of Artemia fed with different feeds and inoculated with different dead or live bacterial strains were investigated with analysis of variances and multiple comparisons of Tukey's range. All statistical analysis was tested at a 0.05 level of probability using the software Statistica 5.5 (Statsoft, Inc.).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of bacterial strains.
Three strains included in the present study, i.e., LVS 2, LVS 8, and a representative of cluster A, were characterized by blasting their complete 16S rRNA gene sequences in GenBank (Table 1). Strain LVS 2 was found to belong to the genus Bacillus, possibly representing a currently undescribed species that is phylogenetically closest to the marine species Bacillus aquimaris and Bacillus marisflavi. Strain LVS 8 was allocated in the Vibrio splendidus group and was most closely positioned to the species V. tasmaniensis, V. lentus, and V. cyclitrophicus of this group. The representative of cluster A was classified as a member of the -Proteobacteria but could not be clearly assigned a particular genus in this phylogenetic group. Its closest phylogenetic neighbors included the genera Ruegeria and Silicibacter.

Artemia fed with poor-quality feed.
Artemia fed with the poor-quality WT yeast was inoculated with 10 bacterial strains added either dead or live and compared with nauplii fed with the same yeast in the absence of bacteria. Results presented in Table 2 (experiment 1) show that the addition of small amounts of dead bacteria (Table 1) could strongly improve Artemia TBP in comparison to the bacterium-free control, mostly due to an increase in the survival rate rather than a better individual length (IL). LVS 2 was the only strain with which no significant improvements in Artemia performance were observed. The enhanced Artemia performance was, however, highly dependent on the procedure used to kill bacteria. The most remarkable increase in TBP was obtained with -irradiated preparations of strains LVS 3 and LVS 8 and with a norvanol-treated preparation of LVS 8 and GR 10. With other bacteria (i.e., GR 8, GR 11, GR 12, and cluster A), autoclaving supported a higher increase in TBP in comparison to -irradiation, mainly due to significantly higher survival.

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TABLE 2. Average survival, IL, and TBP of Artemia fed with poor-quality WT yeast and inoculated with 10 bacterial strainsa

In terms of live bacteria, significant improvements in Artemia TBP were observed in nauplii inoculated with six strains (LVS 2, LVS 3, LVS 8, GR 8, GR 10, and GR 11) in comparison to bacteria-free nauplii, while the opposite occurred when live Vibrio campbellii strain LMG21363 was added. All differences were due especially to significant variations in survival and to a lesser extent in IL of nauplii. For GR 12, cluster A, and Vibrio proteolyticus strain CW8T2, no significant differences were observed in Artemia TBP. Only two bacterial strains (i.e., LVS 2 and GR 8) increased Artemia performance (although not always significantly) in all tested parameters (survival, IL, and TBP) when live bacteria were added instead of dead bacteria. Yet overall, none of the tested bacterial strains yielded significantly better Artemia TBP when added live in comparison to the equivalent treatments with inactivated bacteria.

Artemia fed with medium-quality feeds.
Results of survival, IL, and TBP with Artemia fed with the medium-quality feeds (mnn9 yeast and the microalga DT CCAP 19/27) and inoculated with 10 bacterial strains added dead or live are presented in Tables 3 (experiment 2) and 4 (experiment 3), respectively. With both feeds, when dead bacteria were provided to nauplii, higher values of TBP were observed in comparison to the bacterium-free control. However, significantly higher performances were observed only in Artemia fed mnn9 yeast and inoculated with dead preparations of LVS 3, LVS 8, Vibrio campbellii strain LMG21363, and Vibrio proteolyticus strain CW8T2, mainly due to significantly higher IL. Nauplii fed with the microalga DT CCAP 19/27 presented significantly higher TBP when supplied with dead LVS 3, LVS 8, GR 8, GR 10, GR 11, GR 12, and cluster A. Artemia fed mnn9 yeast and inoculated with dead bacteria killed with different methods revealed almost no differences in TBP. The only exceptions occurred with the strain LVS 2 (significantly lower performances in nauplii inoculated with autoclaved bacteria) and LVS 3, GR 12, and cluster A (significantly lower performances in nauplii inoculated with norvanol-killed bacteria). Yet nauplii fed with the microalga DT CCAP 19/27 presented, in most cases, higher variability in TBP depending on the procedure used to kill bacteria, except with strain LVS 2. -Irradiated preparations of GR 8, GR 10, GR 11, GR 12, and cluster A enhanced the performance of Artemia in comparison to the other two killing methods. For strain LVS 3, the highest nauplius TBP was achieved with autoclaved bacteria, while for strain LVS 8, the highest values were obtained with a norvanol-killed preparation. All differences were due to a combination of higher nauplius survival and IL but were not always statistically significant.

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TABLE 3. Average survival, IL, and TBP of Artemia fed with medium-quality mnn9 yeast and inoculated with 10 bacterial strainsa

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TABLE 4. Average survival, IL, and TBP of Artemia fed with medium-quality microalga Dunaliella tertiolecta (strain DT CCAP 19/27) and inoculated with 10 bacterial strainsa

With live bacteria and mnn9 yeast as the main feed, significant improvements in Artemia TBP were observed only in nauplii inoculated with three live strains, i.e., GR 8, GR 10, and GR 12, in comparison to bacterium-free nauplii, mainly due to the significantly higher IL of nauplii. For all other bacterial strains, no significant differences were observed in Artemia TBP. In contrast, nauplii fed with the microalga DT CCAP 19/27 showed significant improvements in Artemia TBP when supplied with four live bacterial strains (i.e., LVS 2, LVS 3, LVS 8, and GR 8). These differences were mainly due to both higher nauplius survival (often significantly) and/or IL (not significantly). When mnn9 yeast was used as feed, no live bacterial strains significantly enhanced the performance of Artemia beyond those registered with inactivated bacteria irrespective of the method used. The opposite occurred for LVS 8 and Vibrio campbellii strain LMG21363, due to decreased survival (LVS 8) or to a combination of both IL and survival (Vibrio campbellii strain LMG21363). In contrast, Artemia fed with the microalga DT CCAP 19/27 and inoculated with live cells of strain LVS 2 grew significantly better (higher values of TBP in comparison to nauplii inoculated with inactivated bacteria), mainly due to improvements in survival. The opposite occurred for strains LVS 8, GR 10, GR 11, and GR 12 and cluster A, due to decreased IL.

Artemia fed with good-quality feed.
Ten bacterial strains were tested dead and live in the gnotobiotic Artemia culture fed with a well-performing strain of the microalga D. tertiolecta (DT CCAP 19/6B). According to Table 5, (experiment 4), significant improvements in Artemia TBP were observed for nauplii inoculated with seven dead bacterial strains (i.e., LVS 3, LVS 8, GR 11, GR 12, cluster A, Vibrio campbellii strain LMG21363, and Vibrio proteolyticus strain CW8T2) in comparison to nauplii in bacterium-free conditions, mostly due to a statistically insignificant increase in IL. The comparison of Artemia fed with dead bacteria killed with different methods revealed significant differences in TBP of nauplii (except for LVS 2). -Irradiated bacteria (GR 8, GR 10, GR 11, GR 12, and cluster A) produced the highest Artemia performances, followed by the other two treatments, while for strains LVS 3 and LVS 8, nauplii performed better with autoclaved and norvanol-killed bacteria. These differences were mainly due to increases in IL of nauplii, which were not always statistically significant.

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TABLE 5. Average survival, IL, and TBP of Artemia fed with good-quality microalga Dunaliella tertiolecta (strain DT CCAP 19/6B) and inoculated with 10 bacterial strainsa

Significant improvements in Artemia TBP were observed only in nauplii inoculated with five live bacterial strains (i.e., LVS 3, LVS 8, GR 8, cluster A, and V. proteolyticus), in comparison to bacterium-free nauplii. All differences were mostly due to significantly higher IL. Living cells of strain GR 8 and cluster A enhanced significantly the performance of Artemia beyond those registered with inactivated bacteria irrespective of the method used, while the opposite occurred for strains GR 11 and GR 12, in all cases mostly due to differences in IL.

Bacteria ash-free dry weight content.
In general, cells of live bacteria had higher AFDW contents than dead bacteria (Table 1) (although not always statistically significant). This might have contributed to the considerably higher TBP observed on Artemia fed microalgae DT CCAP 19/27 (supplied with the live strain LVS 2) and DT CCAP 19/6B (supplied with the live strain GR 8 and live cluster A) in comparison to nauplii inoculated with the same dead bacteria. The highest ratio obtained between the amounts of bacteria and yeast or microalgae added to Artemia in the experiments (in terms of AFDW) occurred in nauplii fed WT yeast (0.034 to 0.059), followed by DT CCAP 19/27 (0.016 to 0.028), DT CCAP 19/6B (0.013 to 0.023), and the mnn9 yeast (0.012 to 0.021). These ratios could explain, especially for WT yeast, the significant improvements registered in survival of nauplii when inoculated with bacteria, although the amount of bacterial biomass given was insufficient to significantly enhance the IL. For the other feeds, even with such low ratios, the addition of bacteria could sometimes significantly enhance Artemia performance (Tables 3 to 5).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although WT and mnn9 yeast were cultured under identical conditions, Artemia fed with mnn9 yeast always performed better (Tables 2 and 3), even when bacteria were added. Similar results were obtained by Marques et al. (29, 30) for axenic Artemia fed with the same yeast strains and using a similar experimental setup. This difference can be due to an improvement in yeast digestibility to Artemia caused by the mnn9 mutation. As postulated by Coutteau et al. (11), the digestive tract of Artemia displays low mannase and high ß-glucanase activities, complicating the digestion of yeast cells rich in cell wall-associated mannoproteins, such as WT yeast, and contributing to the proper digestion of yeast cells with reduced mannoprotein content, such as mnn9. As for the two microalga strains cultured in the same conditions, DT CCAP 19/6B always appeared to be a better feed for Artemia (Tables 4 and 5), even when bacteria were added. This difference was previously reported by Marques et al. (30) for axenic Artemia fed with the same algae using a similar experimental setup and is probably related to differences in the nutritional values of the two strains. Although the reason for the difference in feed quality between the four types of feed is still unclear, the experimental setup of the gnotobiotic environment was found to be very suitable for verifying in which way Artemia can benefit from the presence of bacteria against four nutritionally different backgrounds.

Bacteria can play an important role as a direct feed source for herbivorous zooplankton (40) and are reported to contribute to the nutritional value of Artemia feeds by being a major source (directly or indirectly) of proteins, vitamins (e.g., B₁₂), essential amino acids, fatty acids, polyamines, enzymes, and inorganic nutrients (17, 21). Lipids, as well as proteins, are the main nutrients required during the early developmental stages of Artemia, whereas carbohydrates, together with proteins, are more important for juveniles and adults (12). Levels of myristic, palmitic, stearic, oleic, linoleic, and linolenic fatty acids seem to stimulate growth and fertility of Artemia when added to the culture medium (12, 39), while the lack of some vitamins creates severe deficiencies in the metabolism of Artemia (20, 38). The efficacy of Artemia in bioencapsulating bacteria is dependent on the type of bacteria used, time of exposure, and status (live or dead) of the bacteria (14).

It is obvious from the data that dead bacteria were able to promote TBP of Artemia (Tables 2 to 5) independently from the quality of the feed provided. Especially when the major feed type was of low quality (WT yeast), almost all tested bacteria had a significant positive effect on the TBP (Table 6). However, the AFDW data indicate that when WT yeast is the major feed source, the relative proportion of bacteria in the feed offered was high (Table 1), suggesting that the TBP increase was partly due to the amount of bacterial biomass offered. Yet, when the dead autoclaved Vibrio proteolyticus strain CW8T2 and the Vibrio campbellii strain LMG21363 were added, this ratio was rather low (Table 1), while the relative increase in TBP was among the highest. This suggests that in some cases, the increase in Artemia TBP could not be attributed to the microbial biomass added. Further analysis of data in Tables 2 to 5 suggests that dead bacterial biomass affects Artemia survival to a larger extent, particularly when poor- and medium-quality feeds were used. However, dead bacteria do not seem to supply Artemia with enough essential nutrients to improve IL, since only in rare occasions was a significant increase in this parameter observed. This argues against the idea that dead bacteria can be suppliers of essential nutrients, especially when added in small amounts.

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TABLE 6. Effects of supplying Artemia fed yeast (WT or mnn9) or microalgae (DT CCAP 19/27 or DT CCAP 19/6B) with dead or live bacterial strainsa

Performances of Artemia inoculated with bacteria killed with the three different methods revealed significant differences (Table 6). Overall, two distinct patterns could be observed in the present study. In the first pattern, higher performance of nauplii occurred when -irradiated bacteria were added, followed by norvanol- and autoclave-killed bacteria. According to Marques et al. (29), autoclaving destroys more "sensitive molecules" than less destructive methods such as -irradiation. This fact could explain the results obtained in the first pattern. In the second pattern, autoclaved bacteria supported the highest Artemia performance, followed by norvanol-killed and -irradiated bacteria. Despite destroying "sensitive molecules," autoclaving also damages the cell wall of an organism (including bacteria) (33) by thermal denaturation of proteins essential for cell wall rigidity. In this way, the cell wall becomes weaker (24) and digestion of whole autoclaved bacterial cells by Artemia enzymes is improved. The denaturation of proteins also occurs with norvanol (26) and -irradiation, but to a lesser extent (36).

Previous experiments by Verschuere et al. (50, 51) characterized live cells of strains LVS 2, LVS 3, and LVS 8 as performance enhancing, supporting significant improvements in Artemia TBP compared to the axenic control, while the Vibrio proteolyticus strain CW8T2 was typed as a strain with a negative influence on Artemia performance. Similar results were obtained in the present study when low-quality feed (WT yeast) was used to feed nauplii (Tables 2 and 6), except for live Vibrio proteolyticus strain CW8T2, which acted as a neutral bacterium, thus not influencing Artemia performance. It thus seems that this strain is not a primary pathogen to Artemia but rather an opportunistic organism that expresses its virulence only when Artemia is cultured in suboptimal conditions. In fact, in Artemia fed with the good-quality microalga DT CCAP 19/6B, Vibrio proteolyticus strain CW8T2 even behaved as a performance-enhancing bacterium. Likewise, Verschuere et al. (50, 51) used -irradiation to obtain bacterium-free inert feed that was provided to axenic nauplii. However, -irradiation causes some negative effects on the feed quality (e.g., by destruction of essential nutrients) (29), in this way weakening nauplii provided with such feeds.

Live cells of strains GR 8, GR 10, and GR 11 were considered as performance-enhancing bacteria to nauplii fed poor-quality WT yeast, while the live Vibrio campbellii strain LMG21363 was characterized as a bacterium with a negative influence on the performance of Artemia (Table 6), confirming the findings of Soto-Rodriguez et al. (46) that this bacterium is pathogenic to Artemia. When better-quality feeds were provided to Artemia, different live bacterial strains were considered performance-enhancing bacteria. Yet, independently of the feed added to cultures of nauplii, the strain GR 8 was always a performance-enhancing bacterium in a significant way (Table 6), and thus, this strain can be considered as a candidate for further tests on its quality as a probiont. The two pathogens (the Vibrio campbellii strain LMG21363 and Vibrio proteolyticus strain CW8T2) did not express any detrimental effect when added to medium- and good-quality feeds. Previous findings of Burgents et al. (8) and Patra and Mohamed (35) reported protection of shrimp and Artemia, respectively, by yeast supplements against pathogenic Vibrio.

Beneficial or pathogenic effects caused by a bacterial strain in Artemia were reduced or even disappeared when medium/good-quality feeds were used. This could be due to improvements in the Artemia status (health condition) caused by specific characteristics of the feeds either of a nutritional nature, due to the induction of digestive enzymes secreted by the feed, in the same way as previously described for European sea bass supplied with microalgae (9) and baker's yeast (47), or eventually as a result of nonspecific stimulation of the brine shrimp immune response against the bacteria. Mnn9 yeast possesses high levels of ß-glucans and chitin in the cell wall, while the microalga D. tertiolecta is reported to contain high levels of ß-carotene in its cells (1). ß-Carotene (6, 25), chitin (4, 43), and ß-glucans (10, 32) are involved in the optimal function of the immune system in terrestrial and aquatic animals and are known to enhance overall disease resistance and improved health and performance in aquatic organisms (e.g., shrimp and fish). However, further studies are still needed to verify these hypotheses using the present gnotobiotic system.

Previous studies on the effects of bacteria in Artemia (13, 34, 50, 51) did not clarify the mode of action of live bacteria in Artemia assays. In order to separate the nutritional effects of a bacterial strain from any probiotic effect, the performance of Artemia inoculated with -irradiated, norvanol-treated, or autoclaved bacteria (depending on which treatment had the highest effect) was compared to that of nauplii cultured with the same live bacterium. Significant improvements in Artemia TBP were detected when live cells of LVS 2 (DT CCAP 19/27), GR 8, and cluster A (DT CCAP 19/6B) were added in comparison to dead bacteria. The AFDW was generally lower in -irradiated and norvanol- and autoclave-killed bacterial cells than in the same live bacterium (Table 1). Hence, these enhancements could be partly due to a reduction of the bacterial quality and/or the amount of nutrients by -irradiation. In addition, live bacteria are likely to grow in the gnotobiotic environment during the 6-day test, resulting in the recycling of nutrients which are channeled towards Artemia and in an improvement of the overall water quality (51), or, more specifically, in removing toxic metabolic substances that can adversely affect the growth and survival of Artemia, especially when cultured under suboptimal conditions (50). However, improvements observed in Artemia performance when live bacteria were added could also be related to other beneficial effects of bacteria, such as supplying active bacterial enzymes allowing additional digestive abilities in the intestine of Artemia (23, 50) and inducing digestive enzyme secretion of Artemia.

In contrast, Artemia supplied with the other live beneficial bacterial strains did not profit from the extra amount of nutrients delivered by these bacteria in comparison to nauplii provided with the same amount of dead bacterium. However, these bacteria should not necessarily be discarded as probionts, since their probiotic nature may eventually be expressed only in the presence of pathogens, as these bacteria can enhance the nauplius immune response (52) or prevent the proliferation of opportunistic pathogens by competing for available resources (nutrients, space, adhesion sites in the gut or on the surface of Artemia, etc.) or through antagonism (production of toxic or inhibitory substances) (51, 52).

It became clear that in the above-described gnotobiotic environment, the tested microorganisms can be catalogued in different classes: probionts (which have a beneficial and active effect on an organism and/or on the culture medium), pathogens (including opportunistic bacteria that can cause diseases or any other disturbance in an organism), and neutral microorganisms (not causing any marked effects on an organism). In parallel, many microorganisms can be considered as potential sources of nutrients for Artemia (depending on the nutritional requirements and on the accessibility of the nutrients, e.g., thickness or smoothness of the cell wall), improving its general condition in this way. Yet, further research is still ongoing to elucidate the exact mode of action of the observed beneficial and pathogenic effects of bacteria, combining the present approach with challenge tests and other types of analysis (e.g., transcriptome and proteonomic analyses). Finally, experimental trials in all possible host-target combinations are also required before making any critical assessment.

 
The Foundation for Science and Technology (FCT) of Portugal supported this study through a doctoral grant to A.M. (no. SFRH/BD/4831/2001). The Belgian Foundation for Scientific Research (FWO) financed the project "Functional role and characteristics of microorganisms in the larviculture of aquatic organisms: Artemia as preferred test organism" (no. 350230.02) and the project "Nutritional and immunostimulatory characteristics of isogenic yeast mutants in Artemia" (no. 1.5.125.04). G.H. is a postdoctoral fellow of the FWO.

We are also grateful to Luc van Hoorebeke from the Institute for Nuclear Sciences of the Ghent University for irradiating all bacteria needed for this study and to M. Cnockaert for excellent technical assistance in the 16S rRNA gene sequencing work.

 
* Corresponding author. Mailing address: Laboratory of Aquaculture and Artemia Reference Center, Faculty of Bioscience Engineering, Ghent University, Rozier 44, 9000 Ghent, Belgium. Phone: 0032-92643754. Fax: 0032-92644193. E-mail: marques_am{at}yahoo.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, August 2005, p. 4307-4317, Vol. 71, No. 8
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