Feeding the transgenic microalgae containing bovine lactoferricin enhances the survival of fish infected by Vibrio parahenmoly

Microalga, Nannochloropsis oculata, is an important microorganism for feeding fish larvae. We develop a transgenic line of N. oculata that enbles to produce an antimicrobial peptide, bovine lactoferricin (LFB). An algae-codon-optimized cDNA of LFB was fused with a red fluorescent protein (DsRed) reporter and driven by a heat-inducible promoter, which is a heat shock protein 70A promoter combined with a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 ‘ promoter from Chlamydomonas reinhardtii. This construct was transferred into N. oculataele cells by eletroporation. The successful rate of stable transgenesis at least for 15 months was around 0.4 %(2/491) of the examined micoalgal clones. After the heat induction, the transcripts of LFB-DsRed gene was detected in the transgenic algal clone (0829A) by RT-PCR. In addition, protein analysis showed that a band corresponding to the fusion protein of LFB-DsRed was positive in the immunoblotting analysis by using monoclonal anti-Ds Red antibody. When we orally-in-tube fed injected with algae into medaka, then infected by Vibrio parahaemolyticus for 6 hr, we calculated the survival rate after 24 hr infection. Results showed that the surivial rate of medaka fed with recombinant algae (1×10 cells/ per fish) was greatly higher than that of medaka fed with wild-type algae, 91.6% (n=12) versus 0% (n=12), suggesting that the transgenic microalgae, 0829A, can help to enhance the survival of medaka after V. parahaemolyticus infection. Introduction The aquaculture’s products are one of important food resources for humans. To meet the requirement of increasing population, the large-scale and high-density aquaculture systems are developed intensively. But the yield of aquaculture may be declined markedly if fish and shellfish diseases occur. Under this circumstance, chemical antibiotics are commonly employed in the most economic fish culture to prevent the aquaculture’s products from being suffered diseases, except that only large-sized and expensively fish like trout and salmon can be inoculated with vaccine. Subsequently, the antibiotic resistant pathogens may be selected and spread out along with the waste water to cause seriously environmental pollution. Therefore, to develop a safety, effective and inexpensive biological antibiotic for aquaculture is extremely necessary. Bovine lactoferricin (LFB) is a 3,142-kDa peptide derived from the N-terminus of bovine lactoferrin (from Phe17 to Phe41). LFB can be generated by pepsin hydrolysis in the digestive tract (Bellamy et al., 1992a). LFB is an antimicrobial peptide that enables to kill or restrain many pathogens, including Gram-negative bacteria, such as Escherichia coli (Bellamy et al.,1992a) , Proteus vulgaris (Bellamy et al.,1992b), Klebsiella pneumoniae and Pseudomonas aeruginosa (Yamauchi et al., 1993); Gram-positive bacteria, such as Clostridium paraputrificum, Corynebacterium ammoniagenes, Enterococcus faccalis (Bellamy et al., 1993c), Listeria monocytogenes ( Wakabayashi et al.,1992) and Streptococcus bovis (Bellamy et al.,1992b); parasites, such as Eimeria stiedai (Omata et al., 2001), Giardia lamblia (Turchany et al., 1995), Toxoplasma gondii (Tanaka et al.,1995); fungi, such as Aspergillus fumigatus, Penicillium pinophilum (Bellamy et al.,1994); and virus, such as adenovius (Biase et al., 2003), calicivirus (McCann et al., 2003) and cytomegalovirus (Andersen et al.,2001). Since LFB can suppress general kinds of pathogens, LFB is an excellent material for applying on aquaculture. However, LFB is hardly accessible: it is produced only by the hydrolysis of bovine lactoferin under pepsin treatment in native source (Tomita et al., 2002). This limitation can be solved by making LFB be produce by microalgae. Microalgae are potential materials for using as bioreactors to produce the heterologus proteins mainly because microalgae have many advantages: (1) microalgae enable to produce complicate eukaryotic proteins after post-translational modification (Mayfield et al., 2003); (2) although microaglae are eukaryotic, they can be cultured as cheap and fast as bacteria; and (3) microalgae are considered as food safe microorganisms due to they are free from human pathogens and endotoxin (Tara et al.,2005). Nannochloropsis oculata is a marine unicellular microalga, belonging to Class of Eustigmatophyceae, with a spherical or slightly ovoid shape about 2 to 4 mm in diameter. It consists of polysaccharide wall and contains only one chloroplast (Hibbered, 1981). Beacuse N. oculata can grow in wide range of saline concentration (Hirata et al., 1980) and contains high amount of eicosapentaenoic acid (Sukenik et al., 1993), it is an important microorganism for feeding fish larva and for making green-water of aquaculture ponds (Fulks et al., 1991; Lubzens et al., 1995). In addition of the above advantages that general microalgae have, N. oculata can be cultured in an extremely huge scale by using seashore and sunny sea pond only with fertilizer without constraining the limitations of the shortages of freshwater and cultivable land. Unlike yeast and cell line cultures, expensive medium and aseptic manipulation of N. oculata are not required. More over, N. oculata can survival in widespread salinity, so that it can be applied both in freshwater aquaculture and in seawater aquaculture. The feeding experiments also demonstrate that N. oculata enables to reduce blood pressure in hypertensive rats (Seto et al., 1992). Importantly, N. oculata does not generate gamete (Maruyama et al., 1986). Gamete-based contamination of genetically modified organism has not to be concerned. Lastly, transgenic N. oculata is already available due to cryopreservation technique for N. oculata has been well developed (Poncen and Veron 2003, Cwo et al., 2005). Many studies have been reported on microalgal gene transfer (Boynton et al., 1988; Brown et al., 1991; Kumar et al., 2004; Geng et al., 2003; Borovsky 2003; Geng et al., 2003; Mayfield et al., 2003; Sun et al., 2003; Banicki 2004). However, the selection marker all they used to screen the transformants was antimicrobial resistant gene. Using antimicrobial resistant gene to serve as a bioreactor has to concern about the biological safety, especially if the transgenic microalgae are largely cultured in an open area. In this study, we develop an antimicrobial-free gene transfer system for N. oculata by using red fluorescent protein (DsRed) gene, which originates from coral (Discosoma sp), as a selective marker. In order to release the LBF from the LFB-DsRed fusion protein produced by transgenic N. oculata, we reserved two pepsin cleave sites at Phe2-Lys and at Phe26-Met of LFB-DsRed. It made LFB can be released from LFB-DsRed in aquatic organism’ digestive tract by pepsin digestion. On the other hand, the expression of this fusion protein avoids the destroy of bioreactor when LFB were express along (Kim et al., 2006). We created the transgenic N. oculata to expresses this heterogeous LFB-DsRed fusion protein driven by an inducible promoter from Chlamydomonas reinhardtii (Schroda et al., 2000): a heat shock protein 70A promoter (HSP 70A) combined with ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 ‘ promoter (RBCS 2). The detection of Ds-Red monoclonal antibody and fluorescent microscope demonstrate that the transgenic N. oculata can express LFB-DsRed. After bioassay, the medaka that naturally living in fresh water were domesticate in to sea water orally-in-tube fed injected with tansgenic microalgae, then infected by Vibrio parahaemolyticus for 6 hr. the survival rate showed that the medaka fed with recombinant algae (1×10 cells/ per fish) was greatly higher than that of medaka fed with wild-type algae, 91.6% (n=12) versus 0% (n=12), suggesting that the transgenic microalgae, 0829A, can help to enhance the survival of medaka after V. parahaemolyticus infection