Microbial characterization of enriched Artemia sp. at two ... · Artemia sp. is widely used as live...
Transcript of Microbial characterization of enriched Artemia sp. at two ... · Artemia sp. is widely used as live...
24h storage in starvation
• Pathogen loads are believed to be an important factor in fish larvae
survival due to their limited immune system;
• Larvae mortality is often caused by opportunistic bacteria introduced with
the life feed supplied (Planas and Cunha, 1999);
• Artemia sp. is widely used as live food in larviculture, and a known vector
for introducing bacteria into the systems (Makridis et al., 2010);
• Artemia low temperature storage is a methodology to preserve size and
nutritional profile with no scientific data on bacteria proliferation.
F. Soares*, S. Castanho, M. Moreira, A.C. Mendes and P. Pousão-Ferreira Aquaculture Research Station, National Institute for the Ocean and Atmosphere (IPMA), Av. 5 de Outubro, s/n 8700-305 Olhão, Portugal
ü Cold storage (5±1ºC) seems to be also a viable methodology to restrain total bacterial proliferation after the enrichment
process, temperature affected the amount of total bacteria but not their diversity.
ü Bacteria diversity present in the enriched Artemia seems to be related with the enrichment products used, rather than with
the temperature.
• Buller N.B. 2004. Bacteria from fish and other aquatic animals: a practical identification manual, 1st Edition, CABI Publishing, pp.171-176; • Holt J.G. 1994. Bergey's Manual of Determinative Bacteriology, 9th Edition, Williams & Wilkins.; • Høj L., Bourne D.G., Hall M.R. 2009. Localization, abundance and community structure of bacteria associated with Artemia: Effects of nauplii enrichment and antimicrobial treatment. Aquaculture, 293: 278–285; • Kellam S.J. and J.M. Walker. 1989. Antibacterial activity from marine microalgae in laboratory culture. British Phycological Journal 24:191-194;• Makridis P., L. Ribeiro, M.T. Dinis. 2010. Influence of microalgae supernatant, and bacteria isolated from microalgae cultures, on microbiology, and digestive capacity of gilthead seabream (Sparus aurata) and senegalese sole (Solea
senegalensis) larvae. Journal of the World Aquaculture Society 41(5):780-790; • López-Torres M.A., Lizárraga-Partida M.L. 2001. Bacteria isolated on TCBS media associated with hatched Artemia commercial brands. Aquaculture, 194:11-20;• Planas M. and I. Cunha. 1999. Larviculture of marine fish: problems and perspectives. Aquaculture 177:171-190; • Snoussi M., K. Chaieb, R. Mahmoud, and R. Bakhrouf. 2006. Quantitative study, identification and antibiotics sensitivity of some Vibrionaceae associated to a marine hatchery, Annals of Microbiology 56(4):289-293; • Srinivasakumar K.P. and M. Rajashekhar. 2009. In vitro studies on bactericidal activity and sensitivity pattern of isolated marine microalgae against selective human bacterial pathogens. Indian Journal of Science and Technology 2(8):16-23.
INT
RO
DU
CT
ION
BIBLIOGRAPHY
MATERIALS AND METHODS
CONCLUSIONS
ACKNOWLEDGMENTS
DISCUSSION
ü Artemia enriched with GD showed a lower proportion of Vibrionaceae
possibly due to an effect of antimicrobial molecules produced by the
microalgae (Kellam and Walker, 1989; Srinivasakumar and Rajashekhar,
2009);
ü The Vibrionaceae identified are similar to the species found by López-
Torres & Lizárraga-Partida (2000) and Høj et al. (2009), when they´ve
analysed the bacterial community of Artemia;
ü Vibrio alginolyticus was common in the two enrichments, which is in
agreement with Snoussi et al. (2006), that reports this bacteria as the
most common bacteria in Artemia.
Characterization of the
bacteria flora associated with
Artemia sp. after enrichment
and stored at two different
temperatures.
Microbial characterization of enriched Artemia sp. at
two different temperatures and enrichments
GD - GREEN DIETS, (produced by Necton S.A.)
experimental emulsion of several
freeze-dried microalgaes
supplemented with DHA
OBJECTIVE
Enrichment
RP - Red Pepper®, Bernaqua™
commercial product
Ø Trial setup:
Ø Analytical methodology:
RESULTS
Fig. 1. Total bacteria and Vibrionacea present in the different treatments.GD- enriched
microalgae, RP- Red Pepper®,i- initial sample, (-)5ºC, (+) 19ºC. Letters a, b, c and d indicate
different groups with statistical significant differences (Wilcoxon Rank test, P<0.05).
Ø Bacterial quantification
Ø Vibrionaceae identification
GD - Green Diets
RP - Red Pepper
Other bacteria üMoritella marina / Vibrio
aestuarians
üVibrio coralliilyticus
üVibrio vulnificus
üVibrio pelagius biovar. 2
Common bacteria Vibrio alginolyticus
Room with controlled temperature
GD+ 19±1ºC common temperature for
marine fish larvae rearing
GD-
5±1ºC
RP+ 19±1ºC common temperature for
marine fish larvae rearing
RP-
5±1ºC
Room with controlled temperature
Coolin
g tank
Coolin
g tank
Samples were collected:
1. After enrichment (GDi and RPi)
2. After 24h storage (GD+; GD-; RP+ and RP-)
10ml Artemia + Water
Homogeneization
Plating TSA TCBS
CFU´s counting (7 days)
Selection and isolation of bacteria
Biochemical identification of bacteria
(Holt, 1994; Buller, 2004)
Florbela Soares acknowledge Ciência 2008 program (FCT)
429
LARVI ’13 – FISH & SHELLFISH LARVICULTURE SYMPOSIUM
C.I. Hendry (Editor)
Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium, 2013
MICROBIAL CHARACTERIZATION OF ENRICHED ARTEMIA SP. AT
TWO DIFFERENT TEMPERATURES AND ENRICHMENTS
F. Soares*, S. Castanho, M. Moreira, A.C. Mendes, and P. Pousão-Ferreira
IPMA, Aquaculture Research Station, Av. 5 de Outubro s/n, 8700-305 Olhão, Portugal. *Email: [email protected]
Introduction
In aquaculture larvae survival rates are higher than in nature but mortality is still
very important. Pathogen loads are believed to be an important factor in larval
survival given that in this development stage, the fish immune system is still
limited. Larval mortality is often caused by opportunistic bacteria introduced
mainly by the provided life feeds (Planas and Cunha, 1999).
Artemia sp. is widely used as live food in larviculture to feed marine fish and
penaeid shrimps and a known vector for introducing bacteria into systems, de-
spite the enrichment process used (Makridis et al., 2010).
The process of preserving the enriched Artemia at low temperatures is used in
some hatcheries, although no scientific data about microbiological quality are
available. Candeias-Mendes et al. (2011) showed that essential fatty acids can be
preserved during a 12h period at 5ºC and this is a procedure to consider as a way
for reducing labour and costs in hatcheries.
In this work, the bacteria associated with Artemia enrichment was characterized,
after a normal enrichment protocol and after being starved at normal and low
temperature. Two different enrichment products were tested.
Materials and methods
A continental strain of Artemia sp. from the same batch was divided into two
enrichment products per treatment – GD, an experimental emulsion based of
several freeze-dried microalgaes supplemented with decosahexaenoic acid
(DHA) and RP, a standard commercial product Red Pepper®, Bernaqua™. After
the enrichment process, metanauplii from the two enrichments (GD and RP)
were randomly distributed in two groups: the (+) group maintained at usual ma-
rine fish larvae culture temperature (19±1ºC) and the (–) group kept at low tem-
430
perature (5±1ºC). Therefore the trial had a 4×3 experimental design, with 4
treatments – GD-, GD+, RP-, RP+ – kept in triplicate tanks without food.
Artemia for bacteriological analysis were sampled after the enrichments (GDi
and RPi) period and after 24h at two different temperatures. 10ml of Artemia
and water were sampled and homogenized. After, that the homogenate was se-
quentially diluted ten-fold with 1.5% sterile saline solution, and 100 l of each
dilution was spread in triplicate on agar plates. Tryptic soy agar (TSA, Merck)
was used to obtain the total number of aerobic bacteria, and thiosulfate-citrate-
bile salts-sucrose agar (TCBS, OXOID) was used to isolate and count the bacte-
ria. Plates were incubated at 22ºC for 7 days and counting was made at 2 and 7
days after incubation.
The isolates obtained were examined using phenotypic tests. Routine tests for
determining biochemical characteristics of the isolates were carried out accord-
ing with Holt (1994) and Buller (2004).
Non-parametric (Wilcoxon Rank test) statistical analysis was made with IBM™
SPSS Statistics 21.0.
Results and discussion
After enrichment, Artemia with GD treatment had a lower proportion of Vi-
brionaceae when compared with the RP treatment (Fig. 1). This may be the ef-
fect of antimicrobial molecules produced by the microalgae, present in GD com-
position, since phytoplankton species are capable of producing substances that
are toxic to other bacteria (Srinivasakumar and Rajashekhar, 2009) and some
appear to be naturally bacteriostatic (Kellam and Walker, 1989).
After 24h at 19±1ºC, the total number of bacteria and Vibrionaceae cfus (colony
forming units) present in Artemia increased exponentially when compared with
the cold treatment (Fig. 1). Although, in the Artemia kept at 5ºC, the total bacte-
rial decreased and the proportion of vibrionic cfus increased in both treatments.
The fact that the proportion of Vibrionaceae increased in cold condition can be
supported by Høj et al. (2009), who showed that vibrio cells were relatively
more resistant to antimicrobial treatment, namely cold temperatures. These re-
sults indicate that this preservation method can be routinely used at hatcheries
with clear advantage in routine management. This is an improvement in larval
rearing because bacteria introduced by live feed are an important mortality
cause.
Bacterial identification indicates that some specific Vibrionaceae appear in each
treatment. In the GD Artemia, Moritella marina, Vibrio aestuarians, V. coral-
liilyticus, and V. vulnificus were identified, while in the RP Artemia, V. Pelagius
was identified. V. alginolyticus was common in the two treatments, as reported
431
by Snoussi et al. (2006) and this species has been described as a common bacte-
ria of the intestinal microflora in several marine fish species, with some strains
identified as a pathogen of different marine species (Austin et al., 1993). The
Vibrionaceae identified are similar with the species found in other studies (Høj
et al., 2009).
Fig. 1. Total bacteria (dark grey bars) and Vibrionaceae (grey bars) present in the differ-
ent treatments.GD- enriched microalgae, RP- Red Pepper®, i- initial sample, (-)
5ºC, (+) 19ºC. Letters a, b, c, indicate different groups with statistical significant
differences (Wilcoxon Rank test, P<0.05).
Conclusions
In live feed production, cold preservation (5±1ºC) seems to be a viable method-
ology to restrain total bacterial proliferation, regardless of the enrichment.
Species diversity present in the enriched Artemia is related with the different
treatments and V. alginolyticus was common in both treatments.
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432
Candeias-Mendes A., S. Castanho, J. Coutinho, N.M. Bandarra, and P. Pousão-
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Williams & Wilkins.
Høj L.; D.G. Bourne, and M.R. Hall. 2009. Localization, abundance and com-
munity structure of bacteria associated with Artemia: Effects of nauplii en-
richment and antimicrobial treatment. Aquaculture 293:278-285.
Kellam S.J. and J.M. Walker. 1989. Antibacterial activity from marine microal-
gae in laboratory culture. British Phycological Journal 24:191-194.
Makridis P., L. Ribeiro, M.T. Dinis. 2010. Influence of microalgae supernatant,
and bacteria isolated from microalgae cultures, on microbiology, and diges-
tive capacity of gilthead Seabream (Sparus aurata) and senegalese sole
(Solea senegalensis) larvae. Journal of the World Aquaculture Society
41(5):780-790.
Planas M. and I. Cunha. 1999. Larviculture of marine fish: problems and per-
spectives. Aquaculture 177:171-190.
Snoussi M., K. Chaieb, R. Mahmoud, and R. Bakhrouf. 2006. Quantitative
study, identification and antibiotics sensitivity of some Vibrionaceae associ-
ated to a marine hatchery, Annals of Microbiology 56(4):289-293.
Srinivasakumar K.P. and M. Rajashekhar. 2009. In vitro studies on bactericidal
activity and sensitivity pattern of isolated marine microalgae against selective
human bacterial pathogens. Indian Journal of Science and Technology
2(8):16-23.