EFFECTS OF MICROALGAE SUPPLEMENTATION DIETS ON THE
HUMORAL IMMUNE RESPONSE OF GILTHEAD SEABREAM (SPARUS
AURATA) LARVAE
E. Chaves-Pozo1, M. Arizcun1, A. Vizcaino2, R. García-Cubero3, E. Abellán1
Español de Oceanografía, Centro Oceanográfico de Murcia, 30860 – Puerto de
Mazarrón, Murcia (Spain). E-mail: emilia.abellan@mu.ieo.es
2Dpto. Biología y Geología. Universidad de Almería-CEIA, 04120 – Almería (Spain).
3Algaenergy S.A. Parque Empresarial “La Moraleja”. 28108 – Alcobendas, Madrid (Spain).
1Instituto
Introduction
Although larvae are usually the most susceptible stages to a big array of pathogens, the
defence mechanisms in developing fish are poorly known. Before maturation of
lymphoid organs and immunocompetence, innate mechanisms are essential; however,
few studies were conducted to elucidate defense mechanisms at these early
developmental stages (Zapata et al., 1997). Moreover, the administration of vaccines to
larvae is limited by the ontogeny of the immune system since the administration of
vaccines has to be after immunocompetence is attained. In this framework, the inclusion
of microalgae in the dry diet of commercial fish seem to be a worthy strategy to enhance
the disease resistance of larvae. Taking all this into account we have studied the effect of
the inclussion in the diet of two different microalgae in the humoral immune responses
of gilthead seabream larvae.
Material and Methods
Gilthead seabream larvae of 43 days post-hatching (dph) were randomly distributed in
18 aquaria of 170 L (10 larvae/L). The specimens of three independent aquaria were feed
with the same diet and 6 different diets were evaluated. Thus 6 groups were performed:
the control diet group; two group feed with a control diet supplemented with Tetrasemis
suecica (AlgaEnergy product) at 5% or 10%; two groups feed with a control diet
supplemented with Isochrysis galbana (AlgaEnergy product) at 5% or 10% and group
feed with a commercial diet (Sckretting). After 21 or 55 days of the beginning of the
experiment, the specimens (64 or 98 dph of age, respectively) were sampled, washed
three times with sterile PBS, pH 7.2 and homogenised with 1 volume of phosphatebuffered saline. The supernatants were collected, centrifuged twice at 3000 xg for 5 min
and stored at -80 ºC until use. Protease activity was determined as the percentage of
hydrolysis of azocasein by 2 mg/ml of proteinase K (Charney and Tomarelli, 1947).
Lysozyme activity was measured according to a turbidimetric method that uses the lysis
of Micrococcus lysodeikicus for determination of the lysozyme activity using hen eggwhite lysozyme as standard (Ellis, 1990). Serum antibacterial activity was determined by
evaluating the inhibition on the bacterial growth of Escherichia coli curves (Sunyer and
Tort, 1995). The peroxidase activity was measured with a method previously described
(Quade and Roth, 1997). Data were presented as mean ± S.E.M (n =9) and significance
analysed by ANOVA (p<0.05) and Waller-Duncan post-hoc test.
Results and Discussion
The protease activity was slightly affected after 55 days of treatment mainly with
Isochrysis supplemented diet (Fig. 1A). Thus, the diet with 10% of Isochrysis o
increased the protease activity of gilthead seabream larvae, while no statistically
significant differences were observed between the Tetraselmis supplemented diet or the
commercial diet when compared with control. Regarding peroxidase activity (Fig. 1B),
we observed slightly decreased of activity upon 55 days of dietary intake of the diet
supplemented with 5% of Isochrysis and slightly increase with the diet supplemented
with 5% of Tetraselmis, however, no differences were observed neither in the diet
supplemented with 10% of microalgae nor with the commercial diet when compared
with control diet. No differences were observed in the lysozyme activity between the
different diet analyzed (Fig. 1C), while the bactericidal activity was decreased by all the
diet analyzed including the commercial diet when compared with control diet (Fig. 1D).
As far as we are concern, there is only one study deal with microalgae inclusion on fish
larvae diet, in which increased on some cellular immune responses were observed upon
dietary intake of microalgae (Cerezuela et al., 2012). In that sense our data demonstrated
that the inclusion of microalgae in the diet of gilthead seabream larvae modulates the
humoral immune response.
Figure 1. Humoral immune response in gilthead seabream larva homogenates upon 21
or 55 days of treatment with different diet supplemented with 5 or 10% of microalgae
(Isochrysis or Tetraselmis, respectively) compared with control and commercial diets.
(A) protease, (B) peroxidase, (C) lysozyme and (D) bactericidal activities. Data represent
means ± standard error (n=9). Different letters denote statistically significant differences
between the groups according to ANOVA and a Waller-Duncan post-hoc test (P≤0.05).
Acknowledgements
This work was supported by AlgaEnergy. E. Chaves-Pozo thanks to Ministerio de
Economía y Competitividad for her Ramón y Cajal’s contract.
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