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

* fsoares@ipma.pt

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: fsoares@ipma.pt

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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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

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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.