Use of microalgae bioencapsulated in Artemia during the weaning of Senegalese sole (Solea senegalensis Kaup) Pavlos Makridis a, ⁎, Cristiana Moreira b , Rita Alves Costa a , Pedro Rodrigues b , Maria Teresa Dinis a a Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, Faro, Portugal b University of Porto, ICBAS and IBMC, Rua do Campo Alegre n° 823, Porto, Portugal abstract article info Article history: Received 5 December 2008 Received in revised form 3 March 2009 Accepted 15 April 2009 Keywords: New species Fish microbiota Immunostimulants Gene expression Microbial control The microalgae species Chlorella minutissima and Tetraselmis chuii were bioencapsulated in Artemia metanauplii and used during the weaning of Senegalese sole, Solea senegalensis post-larvae. We evaluated the effect of microalgae on: (i) survival and growth of the Senegalese sole post-larvae and juveniles, (ii) expression of genes related to the non-specific immune system, and (iii) the microbial load of sole juveniles. The survival of sole juveniles at the end of the experiment was significantly higher in the treatments added microalgae in their diet compared with the control treatment (P b 0.05), while there was no effect on the final weight of sole juveniles. The total numbers of bacteria as determined by the counts on Zobell's medium were significantly lower in both treatments added microalgae compared with the control treatment (P b 0.05), whereas no significant differences were shown among the different treatments in the numbers of presumptive Vibrio as determined by counts on TCBS. Four partial nucleotide sequences of genes related to the immune system were isolated from lymphoid tissues of S. senegalensis: natural resistance associated macrophage protein 1 (Nramp1), complement C3, transforming growth factor β1 (TGF-β1), and transferrin. The expression of these four genes was calculated at the end of the experiment in relation to the expression of β-actin, a housekeeping gene. No significant differences (P N 0.05) were determined among the three treatments in relation to the expression of the four genes in homogenates of fish. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Senegalese sole, Solea senegalensis, is a promising new fish species for the aquaculture industry in Southern Europe. It shows low mortalities during the larval stage and a high growth rate in culture (Dinis et al., 1999). Nevertheless, high mortalities have been observed during the weaning of post-larvae to an artificial diet (Fernandez-Diaz et al., 2001; Imsland et al., 2004). Nutritional, microbiological, and zootechnical factors, or a combination of these factors, could be critical for the improvement of the rearing process in order to increase survival rates and predictability of the production process during the weaning stage. Microalgae have a known antibacterial and possible immunostimu- lant effect, as the incorporation of microalgae in the diet increased disease resistance in previous studies (Austin et al., 1992). The anti- microbial effect of microalgae could be caused by bacteria associated with microalgae or by the microalgae cells (Austin et al., 1992; Makridis et al., 2006; Tendencia and dela Pena, 2003). Bioencapsulation of microalgae in Artemia metanauplii after a short-term incubation results in decreased microbial load and a change of species-composition of the bacterial flora associated with the metanauplii (Makridis et al., 2006; Makridis et al., 2000). In addition, microalgae encapsulated in Artemia will eventually be digested by the larvae, provide some additional nutritional value to the Artemia metanauplii as food for the fish larvae, and may have some positive effect on the fish immune system. Live food organisms, such as rotifers and Artemia are applied in the rearing of larvae of marine fish species during the first weeks of feeding, while fish larvae are adapted to be fed with artificial diet during the weaning stage. In the rearing of sole, the weaning may take place either by abrupt, or by gradual replacement of Artemia metanauplii with artificial diet (Canavate and Fernandez-Diaz, 1999; Engrola et al., 2007). In most marine fish species, the specific immune system becomes functional several weeks after hatching, which means that defence against disease in first feeding larvae and post-larvae is based on the non-specific immune system (Falk-Pedersen, 2005; Magnadottir et al., 2005). A preliminary study on the development of the immune system in Senegalese sole has indicated that specific immunity mechanisms are poorly developed during the first weeks after hatching (Castro Cunha et al., 2008). The non-specific system, which includes innate immune Aquaculture 292 (2009) 153–157 ⁎ Corresponding author. Institute of Aquaculture, Hellenic Center for Marine Research, P.O. Box 2214, Heraklion Crete, Greece. Tel.: +30 2810 337762; fax: +30 2810 337778. E-mail address: Makridis@her.hcmr.gr (P. Makridis). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.04.016 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online