To be profitable, aquaculture must optimize rearing conditions to grow fish in as little space and at as fast a rate as possible; however, this high-density rearing condition creates a stressful environment not suitable for several physiological functions of fish. Thus, preventive strategies to control the health and immune responses of the fish play a crucial role in aquaculture.
AMPPs can be used as sensitive indicators of fish health . They are mainly present at sites of potential entry of pathogens and their levels vary significantly even before an animal shows signs of disease, making it possible to intervene before adverse conditions cause overt disease and subsequent loss.
HLP1 and 2 are the most known AMPPs in fish and from the literature we could deduce that, given their ubiquity, they can carry out functions related to defense mechanisms in most teleosts , including D. labrax. Regarding Hb-LP s, it has been shown that their cytosolic breaking can generate peptides that possess biological activity related to hypothetical, secondary functions in different tissues, such as temperature regulation, antibacterial and inflammatory activity, blood pressure regulation, and enhancement of cholinergic transmission . Dicentracin, recently isolated in D. labrax, has a role in immune defense. Using in situ hybridization assay, Salerno et al.  observed dicentracin expression in 68-71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes, whereas dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity.
The predicted 3D structure of the AMPPs in our study is dominated by ordered alpha helices. Most AMPP molecules are frequently alpha-helical, with an overall positive charge (generally +2 to +9) imparted by the presence of multiple lysine and arginine residues and a substantial portion (30% or more) of surface hydrophobic residues. These features seem to play an essential role in the various mechanisms of membrane permeabilization, which often lead to microbial cell death . Microbial killing is a consequence of the interaction of the AMP with the microbial outer membrane, which destabilizes the membrane and promotes channel formation. A first step in this mechanism of action is the electrostatic interaction between the cationic peptide and the negatively charged components of the membrane of the pathogen; hence, an increase in positive charge of the peptide will increase microbicidal activity .
However, membrane permeabilization, per se, does not invariably result in bacterial death. Although AMPs interact with microbial cell membranes, creating pores that lead both to leakage of ions and metabolites, and depolarization, there is increasing evidence to indicate that these effects are not the only mechanisms of microbial killing, and that antimicrobial peptides have other intracellular targets . Some peptides must cross the cytoplasmic membrane and they have developed unique mechanisms to translocate to the cytoplasm. Once in the cytoplasm, translocated peptides can inhibit nucleic-acid synthesis, alter the cytoplasmic membrane septum formation, inhibit cell-wall synthesis, inhibit protein synthesis or inhibit enzymatic activity. An example is buforin II, a histone H2A-derived antimicrobial peptide, which inhibits the cellular functions of E. coli by binding to DNA and RNA after penetrating the cell membranes.
PR-39, a proline/arginine-rich antimicrobial peptide, is able to bind the cell membrane of E. coli without affecting its integrity and kill bacteria by blocking both DNA and protein synthesis. Tachyplesin binds in the DNA minor groove. At their minimal inhibitory concentrations, pleurocidin and dermaseptin both inhibit nucleic acid and protein synthesis without damaging the E. coli cytoplasmic membrane. E. coli cells exposed to these peptides are unable to undergo cell division due to the blocking of DNA replication or the inhibition of membrane proteins that are involved in septum formation [20, 71].
In fish, the tertiary structure of at least two AMPPs of the group of "piscidins" has been resolved by crystallography: [piscidin 1: Protein Data Bank entry 2O JM, and hepcidin: PDB entry 3 HOT http://www.pdb.org/pdb/home/home.do]. Piscidin 1, isolated from hybrid striped bass (Morone saxatilis x M. chrysops), is very similar to dicentracin from sea bass (Dicentrarchus labrax). According to Campagna et al.,  which determined the three-dimensional structure of piscidin, the number of positively charged residues (two arginines, one lysine, and four histidines), and the ability to form an amphipathic helical structure in membrane mimicking environments, are the two main features responsible for the antimicrobial activity of this AMPP. Furthermore, the substitution of two glycine residues, Gly8 and Gly13, with Ala or Pro on piscidin's structure decrease the bacterial cell selectivity of this antimicrobial peptide . In particular, the antimicrobial and haemolytic activities, and the ability to permeabilize the model phospholipid membranes, were higher in piscidin with Gly or Pro at position 8 than for its counterparts with either Gly or Pro at position 13 .
The other peptide of the "piscidin" group, hepcidin, reveals a distorted beta-sheet shape with a hairpin loop. The beta-sheet structure is stabilized by disulphide pairing of Cys residues and hydrogen bonding between the two antiparallel strands . This leads to a markedly amphipathic peptide structure, a hallmark of many antimicrobial and antifungal peptides. The hepcidin 3D conformation is important for its aggregation and function. In particular the proximity of the loop portion of the peptide to the rest of the peptide is a feature associated with the aggregation of the peptide, whereas the rare vicinal disulphide pairing in the hairpin loop may be a significant characteristic in the function of this peptide .
The tertiary structure of other groups of AMPPs such as "Hemoglobin like" and "Histone-like" have not yet been resolved by crystallography in fish. Thus, the structures of Hb-by crystallography in fish. Thus, the structures of Hb-LP, HLP1, and HLP2 were "de novo" predicted in our study. These structures shows a distribution of hydrophobic residues at the surface, suggesting a functional involvement of such residues. This is similar to the aforementioned 3D structures of piscidin 1, and hepcidin. Indeed, sea bass Hb-LP and HLP1 align partially to the alpha helix of piscidin 1, whereas HPL2 aligns partially to the chain C of hepcidin. Furthermore, the charge distribution of the aligned fragments is very similar, suggesting similar functional properties associated to the 3D structure of these AMPPs.
Antimicrobial peptides of animal origin display different types of post-translational modifications that can modify their activity in a significant way; among the most frequent, are glycosylation, and phosphorylation. The analysis of the sea bass AMPP sequences show the presence of O-GalNAc glycosylation sites in HPL1, whereas a motif allowing the N-glycosylation is present in the Hb-LP.
Aside from its important role in antimicrobial activity, little is known about other roles of glycosylation in the lethal mechanisms of the corresponding peptides, though some ideas have been advanced, such as protection against proteinases, modification of secondary structure inhibition of enzymes involved in peptidoglycan biosynthesis or specific recognition between pathogen and peptide . Phosphorylation has been described for histatins although absence of phosphate does not preclude candidacidal activity of this antimicrobial peptide. Other AMPPs such as chromacin, requires both O-glycosylation and tyrosine phosphorylation for full antibiotic activity, and the synthetic nonmodified peptide is completely inactive. Enkelytin, an antibacterial peptide derived from proenkephalin A has two phosphoserines and an oxidized methionine required for activity .
After isolating the sea bass cDNAs of HLP1, HLP2, and Hb-LP, we quantified the expression of these genes and of that of dicentracin (previously isolated by Salerno et al. ) by using real-time RT-PCR in the skin, gills, eyes, stomach, and proximal intestine of three groups of animals. The first group represented the control conditions; the second one was subjected to acute confinement/crowding stress; and the third one was subjected to the same acute stress followed by 24 hours of recovery. Reverse transcription-real-time PCR, which is based on two sequential reactions -- reverse transcription of the mRNA, followed by applying the resulting cDNA as a PCR template -- is considered as one of the most accurate methods for transcript evaluation . In aquaculture, the use of real-time PCR has recently expanded, in particular for detecting microbes, parasites, and genetically modified organisms. However, any need for fast and precise measurements of small amounts of nucleic acids in fish represents a potential niche for real-time PCR-based applications, and as machines become faster, cheaper, smaller, and easier to use, more in-field application needs for this technology in aquaculture are likely to be filled.
The results obtained for the Hb-LP gene showed an increase in the number of mRNA copies in gills and skin of stressed fish as compared to control animals. During the recovery phase, the mRNA copy number of this Hb-LP tends to decrease in both tissues, reaching the control values after 24 hrs of recovery only in skin (Figure 2). Hb-LP expression observed in the gills and skin was higher than in eyes, stomach, and gut. The upregulation of the transcription could be considered as a response for coping with acute stress and it could be associated with an increase in Hb-LP protective activity against noxious external agents. An increase in the antibacterial activity of a hemoglobin-related AMPP, the hemoglobin-like protein 1 (HbβP-1), has been observed in gills and skin of catfish (I. punctatus) after exposure to ectoparasite I. multifillis, and subsequent studies  showed that HbβP-1 is active against another ectoparasite, Amyloodinium ocellatum. This microorganism is also the target of the histone-like protein isolated from the gills of rainbow trout (O. mykiss), catfish, and sea bass [29, 30]. We could deduce that HbβP-1 and its related peptides may act in association with other AMPPs, such as histone-like proteins, isolated from the same tissues, creating a powerful line of innate defense against pathogens. No variation in transcript levels in the other analyzed tissues might indicate that this gene is constitutively expressed and performs its function in immune defense proactively against a stressful event. However, other antimicrobial molecules might play immunological roles in the eyes, stomach, and gut; in fact, a single organism can possess different classes of AMPPs and several variants of the same class . The induction of transcription of Hb-LP gene after four hours of crowding stress, compared to the control samples, is consistent with the data of Liu et al. , who showed that the induction of β-globin requires 3.5 to 24 hours in the presence of interferon or lipopolysaccharide.
The results obtained for HLP1 and HLP2 confirmed that the acute crowding stress induces AMPP transcription and the recovery phase does not always restore mRNA levels to the control values (Figure 3). Thus, the histone-like proteins may also be involved in the immediate early response of the innate immune system. Both HLP1 and HLP2 were expressed mainly in the gills and skin, where a statistically significant change in the number of mRNA copies was observed between control and stressed animals. HLP2 expression was also relevant in eyes, organs that may be damaged by wounds and traumas under conditions of high livestock density. The expression of HLPs in the other tissues, such as gut, could be constitutive, as mRNA levels did not change following acute stress; however, the antimicrobial action in those tissues might be performed by other AMPPs that work together with those analyzed in this work.
Richards et al.  reported that HLP2 isolated from the gut, stomach, and liver of Atlantic salmon (S. salar) was involved in specific immune responses. The authors showed that this protein has antimicrobial activity against Escherichia coli D31, carrying out its functions through active cell secretion during infection events. HLP2 shows antibacterial activity towards both gram-positive and gram negative agents, including Pseudomonas aeruginosa, and anti-parasitic activity against the ectoparasite A. ocellatum, as demonstrated by incubating gill cell lines with the spores of this microorganism 
HLP2 also binds the lipopolysaccharide (LPS) [80, 81], protecting cells from an endotoxic shock mediator, the tumor necrosis factor , and other inflammatory mediators . All these features, combined with increased transcript levels in our experiment, confirm that HLP2 has a central role in modulating the immune system response as a proper immune effector.
HLP1 shows specific antimicrobial activity towards different microorganisms, such as Micrococcus luteus, Bacillus megaterium, E. coli, and Candida albicans[84–86]. Cho and colleagues  have highlighted the role of HLP1 as immune modulator in catfish, where it is present in the epithelial cells in a nonacetylated form. They showed that, after skin injury, HLP1 is proteolysed by cathepsin D to increase the levels of antimicrobial peptide parasin I. Contrary to effects caused by acute stress, chronic stress decreases the transcriptional levels of HLP1 in catfish before any signs of illness can occur. The downregulation of AMPPs transcripts may dramatically increase the susceptibility of fish to infectious diseases . In support of our data, it has been shown that, in rainbow trout, acute stress, such as confinement for a short time (minute to hours), rapidly increases the concentration of plasmatic proteins and stimulates the innate immune system, rather than suppressing it, in order to protect the fish from possible injuries .
Dicentracin gene showed an increase in the number of mRNA copies in skin and gills after the acute stress, compared to the same tissues of control animals. The recovery phase restored transcript levels to the control values only in the skin (Figure 1). The higher expression of dicentracin gene in gills and skin suggests that this AMPP represents a first and immediate line of defense in combating pathogens and stressors since these tissues constitute the first physiological barriers of the animal. Furthermore, in anticipation of a stress, which often occurs during many routine farm practices (hauling, grading, etc.) the AMPP levels in the population would be upregulated so that immune defenses are stronger after the stress is imposed. Under this scenario, the population remains resistant to pathogens commonly present in a latent state in the population. However, we cannot extensively discuss the potential role of the increase in dicentracin mRNA levels based on the results of the present study as we do not know whether such an mRNA profile is consistent with the functional protein levels. Therefore, without protein data we cannot draw any conclusions about the relationship between the mRNA levels and the dicentracin antimicrobial activity in gills and skin of stressed sea bass. Thus, our hypothesis that dicentracin has a role in the acute stress response in fish must be confirmed by further proteomic investigations.