Elimination of the associated microbial community and bioencapsulation of bacteria in the rotifer Brachionus plicatilis
ABSTRACT The bioencapsulation of live bacteria in the rotifer Brachionus plicatilis was determined under monoxenic conditions. The first objective was to evaluate the microbiota of the rotifer during intensive production and to obtain sterile rotifer cultures starting from adult females or amictic eggs using PVP-Iodine, Hydrogen peroxide or antibiotic mixtures. In the rotifers, the proportion of vibrios increased significantly during the mass production, displacing other unidentified marine bacteria. Rotifers, in the absence of culturable bacteria were obtained starting from amictic eggs and using Trimetroprim-sulfametoxasole (Bactrim Roche) at 10 ml l–1. The effect of members of Vibrionaceae on the survival and growth rate of rotifers was determined under monoxenic conditions. The survival of rotifers was not affected in the presence of different isolates, while amictic egg formation occurred and the populations increased when the strains Vibrio proteolyticus C279 and Aeromonas media C226 were tested. All isolates were successfully incorporated in the rotifers, since there was no significant difference between the numbers of bioencapsulated cells of different strains of isolates. The results show that it is possible to replace the microbial community in rotifer cultures, started from disinfected amictic eggs, with selected bacterial strains. This could be used as a tool for future studies to reveal the role of specific bacteria on first larval stages of marine fish species.
- SourceAvailable from: Patrick Sorgeloos[Show abstract] [Hide abstract]
ABSTRACT: Rotifer growth performance and microbial community changes associated with rotifer cultures were monitored while different feed types (Nannochloropsis oculata paste and the commercial yeast based feed CS-3000), different regimes (daily changes, changes per batch and no changes) and mixtures of three probionts (Phenylobacterium sp.; Gluconobacter sp. and Paracoccus denitrificans) were provided. It was shown that the dominant bacterial species in the cultures receiving either N.oculata or CS-3000 were different. However, in cultures receiving both feeds (either switching between feeds on a daily basis or on a batch basis), a high similarity in microbial community fingerprint was found. The presence of probionts was detected by the end of four batch culture cycles in spite of strong shifts of the bacterial community. By group discriminant analysis, it was found that Phenylobacterium sp. and Paracoccus sp. contributed positively to the CS-3000-fed group, while Gluconobacter sp. contributed positively to the N.oculata-fed group, although they did not appear as very dominant species.Aquaculture International 04/2009; 17(4):303-315. · 1.04 Impact Factor
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ABSTRACT: To evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena. Gnotobiotic B. plicatilis sensu strictu, obtained by hatching glutaraldehyde-treated amictic eggs, were used as test organisms. Challenge tests were performed with 11 V. harveyi strains and different quorum sensing mutants derived from the V. harveyi BB120 strain. Brominated furanone [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] as a quorum sensing inhibitor was tested in Brachionus challenge tests. Some V. harveyi strains, such as strain BB120, had a significantly negative effect on the Brachionus growth rate. In the challenge test with MM77, an isogenic strain of BB120 in which the two autoinducers (HAI-1 and AI-2) are both inactivated, no negative effect was observed. The effect of single mutants was the same as that observed in the BB120 strain. This indicates that both systems are responsible for the growth-retarding (GR) effect of the BB120 strain towards Brachionus. Moreover, the addition of an exogenous source of HAI-1 or AI-2 could restore the GR effect in the HAI-1 and AI-2 nonproducing mutant MM77. The addition of brominated furanone at a concentration of 2.5 mg l(-1) could neutralize the GR effect of some strains such as BB120 and VH-014. Two quorum sensing systems in V. harveyi strain BB120 (namely HAI-1 and AI-2-mediated) are necessary for its GR effect on B. plicatilis. With some other V. harveyi strains, however, growth inhibition towards Brachionus does not seem to be related to quorum sensing. Interference with the quorum sensing system might help to counteract the GR effect of some V. harveyi strains on Brachionus. However, further studies are needed to demonstrate the positive effect of halogenated furanone in nongnotobiotic Brachionus cultures and eventually, in other segments of the aquaculture industry.Journal of Applied Microbiology 08/2007; 103(1):194-203. · 2.20 Impact Factor
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ABSTRACT: The purpose of this paper was to investigate the nature of the effect of l-carnitine on the cultures of the marine rotifer Brachionus “Cayman” and its microflora. Xenic and gnotobiotic experiments were set up with rotifers, fed axenic wild-type yeast (Saccharomyces cerevisiae) or xenic microalgae (Tetraselmis suecica) and incubated with l-carnitine concentrations of 0.1, 1, 10, 60, 100 and 1000 mg L−1. Axenic neonates were obtained by separating amictic eggs from the rotifers by blending, prior to disinfection with glutaraldehyde and hatching. The gnotobiotic cultures had a significantly lower growth rate and egg ratio (%), compared to both xenic trials. The xenic cultures fed algae performed significantly better than those fed baker's yeast, when comparing population density, growth rate and egg ratio. A significant effect of l-carnitine addition was only found in yeast-fed cultures. Microbial assays, conducted with similar doses of autoclaved l-carnitine, revealed that several bacterial species present in the community of conventional Brachionus “Cayman” cultures, could possibly utilize l-carnitine for growth. These results suggest that the improvement of population density, growth rate and egg ratio in xenic rotifer cultures supplemented with l-carnitine, is most likely due to the stimulation by this compound of certain species of (beneficial) microorganisms, which are in turn valorized by the rotifers, yielding improved culture performance. Because of the absence of a positive effect under gnotobiotic conditions, a direct effect of l-carnitine on fatty acid metabolism, as suggested in literature, is unlikely.Journal of Experimental Marine Biology and Ecology 01/2010; 393:114-123. · 2.26 Impact Factor
Aquaculture International 11: 95–108, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Elimination of the associated microbial community
and bioencapsulation of bacteria in the rotifer
SERGIO F. MARTÍNEZ-DÍAZ1∗, C.A. ÁLVAREZ-GONZÁLEZ1,
M. MORENO LEGORRETA1, RICARDO VÁZQUEZ-JUÁREZ2and
1Centro Interdisciplinario de Ciencias Marinas IPN, Playa el Conchalito sn. La Paz Baja
California Sur, CP 23060 México;2Centro de Investigaciones Biológicas del Noroeste, La
Paz Baja California Sur. Apdo. Postal 128 CP 23000 México;3Departamento de
Biotecnologías, Universidad Autonoma Metropolitana, Av. Michoacán y la Purísima sn Col
Vicentina 09340 México DF;∗Author for correspondence (e-mail: email@example.com; fax:
Received 20 November 2001; accepted 2 November 2002
Abstract. The bioencapsulation of live bacteria in the rotifer Brachionus plicatilis was
determined under monoxenic conditions. The first objective was to evaluate the microbiota
of the rotifer during intensive production and to obtain sterile rotifer cultures starting from
adult females or amictic eggs using PVP-Iodine, Hydrogen peroxide or antibiotic mixtures.
In the rotifers, the proportion of vibrios increased significantly during the mass production,
displacing other unidentified marine bacteria. Rotifers, in the absence of culturable bacteria
were obtained starting from amictic eggs and using Trimetroprim-sulfametoxasole (Bactrim
Roche?) at 10 ml l−1. The effect of members of Vibrionaceae on the survival and growth
rate of rotifers was determined under monoxenic conditions. The survival of rotifers was not
affected in the presence of different isolates, while amictic egg formation occurred and the
populations increased when the strains Vibrio proteolyticus C279 and Aeromonas media C226
were tested. All isolates were successfully incorporated in the rotifers, since there was no
significant difference between the numbers of bioencapsulated cells of different strains of
isolates. The results show that it is possible to replace the microbial community in rotifer
cultures, started from disinfected amictic eggs, with selected bacterial strains. This could be
used as a tool for future studies to reveal the role of specific bacteria on first larval stages of
marine fish species.
Key words: Bioencapsulation, Gnotobiotic rotifers, Rotifer microbiota, Vibrionaceae
Abbreviations: ATCC – American Type Culture Collection; MA – Marine Agar; OD – Optic
Density; ASW – Autoclaved Sea Water; TCBS – Thiosulfate-citrate-bilis salt-sucrose agar;
CFU – Colony Forming Unities; TSA – Trypto casein soy agar; PVP – Polyvinyl Pirrolidone;
PEC–Penicillin-Streptomycin-Chloranphenicol; TmSx–Trimetoprim-Sulfametoxasole; BHI
– Brain Heart Infusion; Ppt – parts per thousands; ANOVA – Analysis of Variance.
Brachionus plicatilis culture is an indispensable aspect of marine fish produc-
tion. In most species they are used as live feed during early larval stages.
However, during the mass culture of rotifers, a complex bacterial ecosystem
develops and as a consequence the rotifers can be considered as an actual
and important source of bacteria for fish larvae (Verdonck et al. 1994). These
bacteria are an important component for rotifer production, some are used
directly as food by the rotifers, and others, e.g. the vitamin B12-producing
bacteria, can support the growth ofrotifers, or are indispensable forsustaining
rotifer growth when algae or yeast are used as food (Hino 1993; Yu et al.
1988; Yu et al. 1989; Yu et al. 1990b; Hirayama and Maruyama 1991; Hagi-
wara et al. 1994). As part of the first food, the rotifer-associated bacteria
modify the gut flora present during early larval stages (Muroga et al. 1987;
Nicolas et al. 1989). In nutritional terms, the bacteria present or added in
the rotifer cultures can improve the dietary value of rotifers for fish larvae
(Gatesoupe 1991b; Gatesoupe et al. 1989); however, the rotifer-associated
bacteria could be detrimental to larval performance and survival (Gatesoupe
1982; Gatesoupe 1989; Perez-Benavente and Gatesoupe 1988).
Although it has been reported that a closed relationship exists between
the incidence of opportunistic pathogens and the mass mortalities during the
larval rearing, only in few cases have the etiologic agent and the mechanisms
of bacterial pathogenesis been elucidated e.g. Vibrio sp. and Vibrio anguil-
larum (Masumura et al. 1989; Grisez et al. 1996). A necessary step in order
to clarify the etiologic agent or the mechanisms of bacterial pathogenesis
in larvae is the adaptation of an experimental model to induce infection
under controlled conditions. During the last decade, bacteria-loaded live
feeds have been used as vectors for delivering vaccines (Campbell et al.
1993) and probiotics (Gatesoupe 1990) and also can be used as biocapsules
during experimental infections through oral challenge in larvae (Munro et
al. 1995). In that case it is desirable to eliminate the interference that the
rotifer-associated bacteria could have during the infection. For this reason,
several strategies have been described to reduce or modify the bacterial load
of the rotifers prior to being used as food or biocapsules e.g. rigorous washing
together with freshwater baths and starvation periods (Planas and Cunha
1999), disinfection of resting eggs (Douillet 1998; Rombaunt et al. 1999)
or adults (Munro et al. 1999), the use of antibiotics in adults or amictic eggs
(Perez-Benavente and Gatesoupe 1988; Munro et al. 1995) and incubation
in bacterial suspensions (Gatesoupe 1990; Makridis et al. 2000). The present
study was conducted as a first approach to quality the rotifers as biocapsules
for oral challenge in fish larvae.
The aims of this study were (i) to estimate the number of culturable
bacteria in rotifers during their culture (ii) to evaluate two disinfectants and
two antibiotic mixtures to eliminate the bacterial load of rotifers and (iii) to
estimate the pattern of bacterial bioencapsulation in gnotobiotic rotifers.
Materials and methods
Samples of Brachionus plicatilis were obtained form the UPIMA-
experimental Hatchery (La Paz B.C.S. México). The strain was isolated
from the San Pedrito oasis B.C.S. México and was acclimatized to marine
conditions (Rueda Jasso 1996). Samples were taken from the stock culture
19-l vessels and from the 300-l mass production tanks kept under the usual
rearing conditions for this hatchery. The rotifers were maintained under
asexual reproduction, and each day the rotifers were sieved and resuspended
in filtered seawater adjusted to 25 × 106cells ml−1of the microalgae Nanno-
chloris sp. Aeration was continuous and lighting was supplied from 55-W
fluorescent lamps. Temperature was maintained at 24 ± 2◦C.
Bacteria and culture conditions
The strains Vibrio carchariae ATCC35084, Vibrio campbellii ATCC25920,
Vibrio parahaemolyticus ATCC14126 and the local isolates Aeromonas
media (C281), Vibrio carchariae (C280), Vibrio proteolyticus (C282),
Aeromonas ichtiosmia (C302), Vibrio carchariae (C303) were used as
target bacterium during the present study. The strains C281, C280, C282,
C302 and C303 were isolated during routine seed production in the facili-
ties of CICIMAR, Baja California Sur, Mexico. The isolated strains were
presumptively identified using BIOLOG GN2 microplates (microplates
Biolog, Hayward, CA, USA). In the Biolog test, bacteria were pre-cultured
on tryptic soy agar plates supplemented with 2.5% NaCl, and the subsequent
test was carried out following the procedure provided by the manufacturer.
The strains were maintained in culture tubes at 15◦C. For each experiment a
sample of each bacteria was taken from the maintenance tube and inoculated
on plates of marine agar 2216 (MA). The plates were incubated at 30◦C
during 24 h and the resulting biomass was washed twice in sterile saline
solution (2.5% NaCl). The density was adjusted to an optical density of 1
at 550 nm (OD550= 1) (approx. 109cells ml−1) using a spectrophotometer
SQ118 (MERCK). The number of viable cells in each adjusted suspension
was estimated by inoculating decimal dilutions on MA plates according to
standard microbiological procedures. The final concentration used during the
bioencapsulation experiments were: 5.5 × 107ATCC 35084 ml−1, 4.6 × 107
ATCC25920 ml−1,5.1×107ATCC17802 ml−1,6×107ATCC15338 ml−1,
5.4 × 107ATCC 14126 ml−1, 6.2 × 107C281 ml−1, 6.5 × 107C280 ml−1,
4.6 × 107C282 ml−1, 5.5 × 107C302 ml−1and 6.1 × 107C303 ml−1.
The rotifer-associated bacteria were evaluated in samples from the stock
cultures (19-l carboys) and from the mass production unit (300-L). The
samples were taken over a year long study. 5.0 ml of rotifers were collected
on a 35 µm sieve and washed with 50 ml of autoclaved seawater (ASW).
100 rotifers were homogenized in 5 ml of ASW using a tissue homogenizer.
A tenfold dilutions of the homogenized rotifers were inoculated on plates of
MA in triplicates, Thiosulfate-citrate-bilis salt-sucrose agar (TCBS; Difco)
andMacConkey (Difco) and incubated at30◦Cfor 24 h.Using the plates with
ca. 300 colony forming units (CFU), three representatives of the numerically
most abundant morphotypes were isolated on plates of trypto casein soy agar
TSA (Difco). The isolates were identified at genus level using the identifica-
tion keys of Muroga et al. (1987). The results were expressed as percentages.
However, because the selectivity of the procedure the percentages apply only
to non-randomly selected culturable bacteria.
Elimination of the rotifer-associated bacteria
Two disinfectants and two antibiotic mixtures were tested in order to obtain
bacteria-free rotifers. The evaluation was done using adult rotifers and
isolated amictic eggs as follow:
From adult rotifers
The rotifers were collected in a 35 µm sieve and twice washed with 500-
ml sterile seawater. The rotifers were distributed in individual 35 µm sterile
sieve at an approximate density of 3000 rotifers sieve−1. Each sieve with
rotifers was submerged separately in triplicate in the following disinfectant
solutions: 1) Polyvinyl Pirrolidone-Iodine (PVP-Iodine ISP Technologies) at
0, 0.1, 1, 2, 3, 4, 5, 8, 10 and 15 mg ml−1, and 2) Hydrogen peroxide (Sigma)
at 0, 0.5, 3, 5 and 7% final concentration. In addition, ca. 3000 rotifers were
introduced in triplicate in 10-ml tubes with two mixtures of antibiotics at
different concentrations: 1) PEC (Penicillin 100 mg + Streptomycin 50 mg +
Chloramphenicol 10 mg in 10 ml of sterile seawater) at 0, 20, 40, 60, 180,
200, 300, and 500 µl per tube and 2) TmSx (Trimetoprim + Sulfametoxasole
40 + 8 mg ml−1, Bactrim?, Roche) at 0, 20, 40, 60, 80, 100, 200, and 500 µl
per tube. The experimental controls were the treatments without disinfectant
or antibiotic. The rotifer survival and the relative motility were evaluated
under stereoscopic microscope at 40x magnification. In each treatment, the
antibacterial effect was evaluated using Brain Heart Infusion Media (BHI-
Difco) supplemented with 1.5% NaCl at 1, 3, 5, 10, 30 and 60 min in the
disinfectant treatments and at24 and 48 hrs. in the antibacterial treatments. At
each combination of concentration and time a sample of rotifers was washed
twice with 100-ml autoclaved seawater and then 100 rotifers were homogen-
ized as previously described. Six tubes with 5 ml of BHI were inoculated
with 1 ml of the homogenate. The relative bacterial load was recorded as the
increase in turbidity at 640 nm in a spectrophotometer SQ118 (Merck) after
24 h at 30◦C incubation.
From rotifer eggs
Usually, under normal conditions, rotifer reproduction occurs in an asexual
mode, each female produces amictic eggs, which remain attached to the
mother for some time. In culture each female could have up to 9 eggs attached
at her body depending on the culture conditions.
The amictic eggs were removed from the adult females using a tissue-
tearor (Biospec Product) and washed with abundant sterile seawater at low
temperature (15◦C) in order to retard the hatch. The eggs were dispensed
on sieves and in 10 ml tubes at approximately 3000 eggs by recipient and
treated with equivalent concentrations of disinfectants and antibiotics as used
with adult rotifers. The effect on the bacteria load was evaluated as described
previously for adults.
Effect of selected bacterial strains on the gnotobiotic rotifers
The effect of selected strains of Vibrio and Aeromonas on the gnotobiotic
rotifers was evaluated in 1-l bottles with 0.5 l of 0.2 µm filtered and auto-
claved seawater at 35 ppt. The rotifers (from TmSx eggs treated as previously
described), were added to 100-ml bottles at adensity of 80 rotifers ml−1.Each
flask with rotifers was inoculated with 10-ml of bacterial suspension (OD550
=1) to get afinal concentration of ca. 3.4 ×108cells ml−1.Controls of rotifers
only, bacteria only and algae-rotifer were evaluated simultaneously. For the
algae-rotifer controls, each bottle was adjusted to an initial density of 25 ×
106cell ml−1of a gnotobiotic culture of Nannochloris sp. provided by the
strain collection ofCICIMARLaPaz,México. Eachtreatment was assayed in
triplicate and the experiment was repeated twice. The bottles were maintained
in a water bath at 25◦C. Over 48 h, 5-ml samples were taken from each bottle
at intervals of 6 h under aseptic conditions in order to evaluate the rotifer
density and fecundity. Changes in absorbance at 540 nm from the water were
measured in a spectrophotometer SQ118 (Merck).
Bioencapsulation of selected bacterial in the rotifer Brachionus plicatilis
The procedure used by Gomez-Gil et al. (1998) to bioencapsulate bacteria
in brine shrimp was modified to evaluate the bioencapsulation of bacteria
in the rotifer Brachionus plicatilis. Gnotobiotic rotifers (TmSx treated as
previously described) were added to 250-ml flasks witha bacterial suspension
(sterile seawater and the target bacteria) at a density of 5 rotifers ml−1. The
controls were bacteria and rotifer only. Each treatment was assayed in tripli-
cate. Samples of rotifers from each flask were used to evaluate the number of
bioencapsulated bacteria at 0, 1.5, 3, 6, 12 and 24 hours after the rotifers were
placed in the flask. 20-ml samples from each replicate were collected under
sterile conditions, thoroughly washed, and 100 rotifers were macerated in a
tissue homogenizer. Serial dilutions were prepared, and 100 µl of different
dilutions were spread on plates of MA and TCBS media (Difco). The plates
were incubated at 30◦C for 24 h, and the CFU were counted.
Rotifer associated bacteria
In the stock culture, we found a mean number of rotifer-associated bacteria of
1.4 × 102CFU rotifer−1(n = 15). Numerically document bacteria were other
than Vibrio or Pseudomonas spp. (Table 1). Vibrionaceae occurs in less than
1% of the total isolated bacteria. The other unidentified bacteria were at least
seven different morphotypes characterized on basis of colony morphology,
all Gram(–), catalase(+) and occurs in abundances of 11.7, 14.2, 12.2, 2,
15.5, 2, 8.8%. In 6 of 15 samples we found a Gram-positive Micrococcus-like
bacteria, which was not found in mass production samples.
Differences in the number and taxonomic composition were found in the
rotifer-associated bacteria during the mass production of rotifers (Table 1).
The number of bacteria increased significantly (p < 0.01) to 2.3 × 103CFU
rotifer−1(n = 13). The average number of Vibrionaceae was significantly
bigger than recorded in the stock culture (p < 0.001). We also found an
increase in the variability of the numbers of each group of rotifer-associated
bacteria (Table 1). No significant correlation between the abundance of Vibrio
and Pseudomonas was found in mass production sample (R2= 0.005, n = 13),
neither between the abundance of Pseudomonas and the unidentified bacteria
(R2= 0.533, n = 13), however a significant inverse correlation between the
Table 1. Composition in percentages of the microbiota isolated from the rotifer
Brachionus plicatilis under two different stages of the production. Data are the
average ± standard deviations and maximums and minimum in parenthesis and
apply only to non-randomly selected culturable bacteria. Other bacteria include
all unidentified isolates.
Group Stage of the production
Stock cultureMass production
Pseudomonas32 ± 7.35
19.95 ± 18.89
Vibrionaceae0.44 ± 0.17
23.20 ± 19.03
Other67.5 ± 6.14
56.83 ± 27.78
Total 1.4·102CFU rotifer−1
numbers of Vibrio and the other unidentified bacteria was found (R2= 0.904,
n = 13).
Disinfection of rotifers
The effective dose for PVP-Iodine to eliminate the rotifer-associated bacteria
exposed during 10 min was 5 mg ml−1. All other concentrations tested
proved to be effective in eliminating the rotifer associated bacteria at 30
min of exposure. Unfortunately, the rotifer and the amictic eggs did not
survive at those combinations of concentration and time. Similar results were
found using hydrogen peroxide, where the concentration was high enough
to eliminate the rotifer-associated bacteria (3% during 5 and 10 min). In
order to verify the rotifer tolerance to different concentrations of PVP-I and
hydrogen peroxide the time of survival was recorded during a new set of
experiments, where mortality was defined as the cessation of movement of
the rotifer corona. The maximum times that the rotifers survived in different
concentrations of PVP-I and hydrogen peroxide are shown in Figures 1a
The antibiotic mixtures affected the survival of rotifers less than the disin-
fectant treatments. With TmSx the minimum concentrations at which the
culturable microbiota of the rotifers was completely eliminated were 100 µl
and 500 µl for 24 h and 48 h respectively. Under the microscope, the apparent
motility of the newly hatched rotifers was affected at concentrations higher
than 200 µl of TmSx. Also the survival of the rotifers was not affected at
Figure 1. Time of survival of the rotifer Brachionus plicatilis exposed to different concentra-
tionsof: (a)Polyvinyl PirrolidoneIodine (PVP-Iodine)and (b) hydrogen peroxide. Theshaded
areas in the graph show the effect on bacterial growth at each combination of concentration
and time (darker means more bacterial growth, white is no bacterial growth).
24 hours of exposure to the different concentrations of TmSx tested, however
dead rotifers were found after 48 h of exposure to 100 µl of TmSx and
survivors were not found after 48 h at 500 µl TmSx (Figure 2a).
In the PEC treatments, the survival of rotifers was not affected during the
assay, but the rotifer-associated bacteria were not completely eliminated at
any of the tested concentrations (Figure 2b).
Figure 2. Effect of different antibiotic concentration on the rotifer-associated bacteria and
survival of B. plicatilis. • = Bacterial growth in uncovered tubes, + = bacterial growth in
covered tubes, ? = rotifer survival at 24 h exposition and ? = rotifer survival at 48 h
exposition. For details please see the text.
Effect of selected Vibrio and Aeromonas strains on the survival of
Monoxenic rotifer cultures were used to evaluate the effect of selected
bacterial strains on the survival and growth rate of the rotifers. The popula-
tions of rotifers in the presence of some pure bacterial strains increased
significantly in number after 24–48 h of exposure. The rotifers exposed
Figure 3. Global pattern of bioencapsulation of bacteria in the rotifer Brachionus plicatilis
during a bath challenge. Data are the average of the concentrations registered for different
isolates during independent but similar experiments; Whiskers are the standard error.
to strains Vibrio proteolyticus C279 and Aeromonas media C226 began to
produce amictic eggs approximately 24 hours after the addition of bacterial
cells. After 48 h, the absolute number of rotifers in those treatments increased
from 80 rotifer ml−1to 168 and 126 rotifer ml−1, which represents an increase
of 75% and 32% respectively. The rotifer population in the controls without
bacteria or in the presence of Vibrionaceae C227 and Vibrio proteolyticus
C282 did not grow.
Bioencapsulation of selected bacteria in the rotifer Brachionus plicatilis
Bacteria were successfully incorporated into gnotobiotic rotifers. A similar
pattern between the different bacterial isolates was found; typically, the
number of bacteria per rotifer increased during the first 1.5–3 h, then dropped
to levels near 2000 CFU rotifer−1, stabilizing to 6–24 h (Figure 3). No
significant difference in the number of bioencapsulated cells per rotifer was
found when different strains were used (ANOVA, p > 0.1). Also, mortality
or changes in motility of the rotifers were not observed during these experi-
ments. At 3 h of exposure, the bioencapsulated cells of Vibrio harveyi ATCC
14126 reached values 2.5-fold higher than other strains, however, the number
decreased rapidly at 6 h. The number of biencapsulated cells of Vibrio
carchariae C303 showed a peak at 3 h which was maintained without change
at 6 h, dropped to a minimum level at 12 h and then increased again at 24 h.
The reduction and the control of rotifer associated bacteria is a recom-
mended practice for preventing infection and mortality during fish rearing.
This is necessary because of the risk of opportunistic pathogens such as
Vibrio, Aeromonas and Pseudomonas (Gatesoupe 1990; Makridis et al.
2000). However, apparently the conditions used for the mass production of
rotifers promote the proliferation of these undesirable bacteria. In the present
study, we found that during the mass production of rotifers, the number of
Vibrio increase significantly, displacing populations of other bacterial groups.
Although the natural occurrence of vibrio in the rotifer microbiota can be
questionable, (because the artificial conditions used in the rotifer produc-
tion i.e. salinity, density, water origin, feed); the negative effect of Vibrio in
aquaculture has been widely documented. For example, some Vibrio strains
can produce the collapse of rotifer cultures (Yu et al. 1990a; Hino 1993). In
the present study, some Vibrionaceae strains isolated from rotifer culture or
larval rearing, were evaluated in gnotobiotic rotifers. However, none of the
tested bacteria produced detrimental changes in the rotifer survival, in fact,
the added bacteria were used as food by the rotifers and the recorded increase
in the rotifer populations was supported on a diet of bacteria, suggesting that
the presence of those bacteria in rotifer cultures should be considered benefi-
cial for rotifer nutrition. However, the presence of high numbers of Vibrio in
the rotifers is considered adverse in terms of sanitary control, because several
Vibrio species are opportunistic pathogens for fish larvae. For this reason, it
is desirable to prevent the dominance of Vibrionaceae in rotifer microbiota
In the present study, different strategies to eliminate the rotifer microbiota
were evaluated. It was found that adult females are very sensitive to the
disinfectants PVP-Iodine and hydrogen peroxide, both of which are widely
used in aquaculture. Rotifer mortality was recorded before the elimination of
bacteria could be reached. Similar results were found with amictic eggs when
the eggs lost their viability when exposed to the combinations of concentra-
tion and time necessary to eliminate the epibiotic bacteria of the egg. Also
the use of antibiotics in adult females, only served to diminish but not to
eliminate the associated bacteria. By contrast, the antibiotic treatment applied
to amictic eggs was effective in producing germ free cultures of rotifers. The
best results were obtained at 24 h of exposure to concentrations between 100
and 500 µl of TmSx (equivalent to 10 and 50 ml l−1) when the associated
bacteria were completely eliminated without any effect on rotifer survival,
growth or reproduction.
Germ-free organisms are a valuable tool for studying microbiota-
attributed functions, such as their role in the digestion or in the development
of the immune system. Other applications include the evaluation of the
probiotic potential of selected strains and the study of nutritional require-
ments without the effect of the intestinal microbiota. Also, using germ-free
organisms it is possible to eliminate the undesirable interference of microbial
contaminants during studies of pathogenicity, parasitism or during evalu-
ations oftoxicity. Thiscan occur because some microorganisms compete with
the pathogens or can degrade or modify the toxic substances.
The availability of an adequate vector is a prerequisite for evaluating the
mechanisms of infection through the gastrointestinal tract in larvae i.e. after
feeding with contaminated feeds (Chair et al. 1994), bearing in mind that
infection of marine fish larvae can occur throughout the food chain (Sera and
Kumata 1972; Campbell and Buswell 1983; Muroga et al. 1987). During the
present study the bioencapsulation of bacteria which are potentially patho-
genic tofish wasachieved in gnotobiontic rotifers. Asimilar bioencapsulation
pattern in rotifers was described by Makridis et al. (2000) under non gnoto-
biotic conditions; they found an effective accumulation within 20–30 min,
without detecting differences between strains. In consequence, rotifers are an
adequate oral vector to induce bacterial infections under in vitro conditions.
This work was supported by the National Council of Science and Technology
CONACyT-México. The authors thank Dr F. J. Gatesoupe for critical review
of this paper, Dr. B. Gomez-Gil for providing the reference strains used in
this study, the personnel of the Marine Hatchery of CICIMAR for technical
assistance and Manolo Magaña-Alvarez for editing this English-language
Campbell R., Adams A., Tatner M.F., Chair M. and Sorgeloos P. 1993. Uptake of Vibrio
Anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry.
Fish Shellfish Immunol. 3: 451–459.
Campbell A.C. and Buswell J.A. 1983. The intestinal microflora of farmed Dover sole (Solea
solea) at different stages of fish development. J. Appl. Bacteriol. 35: 215–225.
Chair M., Dehasque M., Van-Poucke S., Nelis H., Sorgeloos P. and De-Leenheer A.P. 1994.
An oral challenge for turbot larvae with Vibrio anguillarum. Aquacult. Int. 2: 270–272.
Douillet P. 1998. Disinfection of rotifer cysts leading to bacteria-free populations. J. Exp. Mar.
Biol. Ecol. 224: 183–192.
Gatesoupe F.J. 1982. Nutritional and antibacterial treatments of live food organisms: The
influence on survival, growth rate and weaning success of turbot (Scophtalmus maximus).
Ann. Zootech. 4: 353–368.
Gatesoupe F.J. 1989. Further advances in the nutritional and antibacterial treatments of roti-
fers as food for turbot larvae, Scophtalmus maximus (L.). In: M. de Pauw, E. Jaspers,
H. Ackfors and N. Wilkins (eds), Aquaculture: A Biotechnology in Progress, Vol. 2.
European Aquaculture Society, Bredene, Belgium, pp. 721–730.
Gatesoupe F.J. 1990. The continuous feeding of turbot larvae, Scophtalmus maximus, and
control of the bacterial environment of rotifers. Aquaculture 89: 139–148.
Gatesoupe F.J. 1991a. Experimental infection of turbot larvae, Scophtalmus maximus (L.),
with a strain of Aeromonas hydrophila. J. Fish Dis. 14: 495–498.
Gatesoupe F.J. 1991b. The effect of three strains of lactic bacteria on the production rate
of rotifers, Brachionus plicatilis, and their dietary value for larval turbot, Scophtalmus
maximus. Aquaculture 96: 335–342.
Gatesoupe F.J., Arakawa T. and Watanabe T. 1989. The effect of bacterial additives on the
production rate and dietary value of rotifers as food for Japanese flounder, Paralichthys
olivaceus. Aquaculture 83: 39–44.
Gomez-GilB.,Herrera-VegaM.A.,Abreu-Grobois F.A.andRoque A.1998. Bioencapsulation
of two different Vibrio species in nauplii of the brine shrimp (Artemia franciscana). Appl.
Env. Microbiol 64: 2318–2322.
Grisez L., Chair M., Sorgeloos P. and 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.
Hagiwara A., Hamada K., Hori S. and Hirayama K. 1994. Increased sexual reproduction in
Brachionus plicatilis (Rotifera) with the addition of bacteria and rotifer extracts. J. Exp.
Mar. Biol. Ecol. 181: 1–8.
Hino A. 1993. Present culture systems of the rotifer (Brachionus plicatilis) and the function
of micro-organisms. In: C.S. Lee, M.S. Su and I.C. Liao (eds), Finfish Hatchery in Asia:
Proceedings of Finfish Hatchery in Asia ’91, Vol. 3. TML Conference Proceedings, pp.
Hirayama K. and Maruyama I. 1991. Vitamin B sub(12) content as a limiting factor for mass
production of the rotifer Brachionus plicatilis. In: P. Lavens, P. Sorgeloos, E. Jaspers and
F. Ollevier (eds), LARVI’91, Vol. 15. pp. 101–103.
Makridis P., Fjellheim A.J., Skjermo J. and Vadstein O. 2000. Control of the bacterial flora
of Brachionus plicatilis and Artemia franciscana by incubation in bacterial suspensions.
Aquaculture 185: 207–218.
Masumura K., Yasunobu H., Okada N. and Muroga K. 1989. Isolation of a Vibrio sp. the
causative bacterium of intestinal necrosis of Japanese flounder larvae. Fish Pathol. 24:
Munro P.D., Barbour A. and Birkbeck T.H. 1995. 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. Env. Microbiol. 61:
Munro P.D., Henderson R.J., Barbour A. and Birkbeck T.H. 1999. Partial decontamination of
rotifers with ultraviolet radiation: The effect of changes in the bacterial load and flora of
rotifers on mortalities in start-feeding larval turbot. Aquaculture 170: 229–244.
Muroga K., Higashi M. and Keetoku H. 1987. The isolation of intestinal microflora of farmed
red seabream (Pagrus major) and black seabream (Acanthopagrus schelegeli) at larval and
juvenile stages. Aquaculture 65: 79–88.
Nicolas J.L. Robic E. and Ansquer D. 1989. Bacterial flora associated with a trophic chain
consisting of microalgae, rotifersandturbot larvae: Influence of bacteria onlarval survival.
Aquaculture 83: 237–248.
Perez-Benavente G. and Gatesoupe F.J. 1988. Bacteria associated with cultured rotifers and
artemia are detrimental to larval turbot, Scophthalmus maximus (L.). Aquacult. Eng. 7:
Planas M. and Cunha I. 1999. Larviculture of marine fish: Problems and perspectives.
Aquaculture 177: 171–190.
Rombaunt G.,DhertPh.,Vandenberghe J.,VerschuereL.,SorgeloosP.andVerstraeteW.1999.
Selection of bacteria enhancing the growth rate of axenically hatched rotifers (Brachionus
plicatilis). Aquaculture 176: 195–207.
Rueda-Jasso R. 1996. Nutritional effect of three microalgae and one cyanobacteria on the
culture of the rotifer Brachionus plicatilis Mükker: 1786. Ciencias Marinas 22: 313–328.
Sera H. and Kumata M. 1972. Bacterial flora in the digestive tract of marine fish. Bacterial
floraof fish, red seabream snapper and crimson sea bream, fed three kinds of diets. Nippon
Suisan Gakkaishi, Bull. Jap. Soc. Sci. Fish. 38: 50–55.
Verdonck L., Swings J., Kersters K., Dehasque M., Sorgeloos P. and Leger P. 1994. Variability
of the microbial environment of rotifer Brachionus plicatilis and Artemia production
systems. J. World Aquacult. Soc. 25: 55–59.
Yu J.P., Hino A., Hirano R. and Hirayama K. 1988. Vitamin B sub(12)-producing bacteria as
a nutritive complement for a culture of the rotifer Brachionus plicatilis. Nippon Suisan
Gakkaishi, Bull. Jap. Soc. Sci. Fish. 54: 1873–1880.
YuJ.P.,HinoA.,UshiroM. andMaedaM. 1989. FunctionofbacteriaasvitaminB12producers
during mass culture of the rotifer Brachionus plicatilis. Nippon Suisan Gakkaishi, Bull.
Jap. Soc. Sci. Fish 55: 1799–1806.
Yu J.P., Hino A., Noguchi T. and Wakabayashi H. 1990a. Toxicity of Vibrio alginolyticus on
the survival of the rotifer Brachionus plicatilis. Nippon Suisann Gakkaishi, Bull. Jap. Soc.
Sci. Fish 56: 1455–1460.
Yu J.P., Hino A., Hirano R. and Hirayama K. 1990b. The role of bacteria in mass culture
of the rotifer Brachionus plicatilis. In: R. Hirano and I. Hanyu (eds), The Second Asian
Fisheries Forum, Proceedings of The Second Asian Fisheries Forum Tokyo. Japan, 17–22
April 1989, pp. 29–32.