The α-helical ascidian peptides include the clavanins and styelins, both groups purified from the haemocytes of Styela clava [25,30]. The four native clavanins are 23-residue, 2.6 kDa AMPs that possess rapid, broad-spectrum antimicrobial activities, inhibiting the growth of Gram-negative (Escherichia coli) and Gram-positive (Listeria monocytogenes) bacteria, as well as the fungus, Candida albicans [25]. Synthetic clavanin A is bactericidal against E. coli and L. monocytogenes at <4 μg mL⁻¹ within 5 minutes of incubation. Clavanins are unusual in that they are histidine-rich and synthetic versions show different modes of antimicrobial activity at different pH values with greater potency under mildly acidic (5.5) pH compared with neutral pH [44]. At pH 7.0 killing is dependent on non-specific membrane disruption but at lower pH the histidines become protonated and membrane disruption probably occurs through interaction with the proteins responsible for generating ion gradients [45]. Clavanins retain their antimicrobial activity at high NaCl concentrations [44].

Styelins were also isolated from S. clava and comprise five 3.7 kDa phenylalanine-rich peptides [30,46]. Two were purified directly from the haemocytes [30] with an additional three members identified by molecular methodologies [46]. One of these, styelin D, was subjected to detailed analyses which reveal that it has potent activity against Gram-positive bacteria (MICs ~5–7 μg mL⁻¹) at pH values from 5.5–7.4 [47]. It is also active against Gram-negative bacteria at pH 5.5 (MIC 2.1 μg mL⁻¹) [47]. Styelins are active at high salt concentrations and the characterization of styelin D reveals that it contains an unusually high number of post-translationally modified residues that are thought to facilitate the antimicrobial activity in high salt and acidic pH conditions [47].

Other ascidian AMPs that form α-helical structures include dicynthaurin, a peptide from Halocynthia aurantium that forms a homodimer consisting of two, 30-residue monomers and has a single cysteine residue [26]. Unusually for an AMP purified from a marine animal, and in contrast to the clavanins and styelins, dicynthaurin is more potently antimicrobial at low salt concentrations, which indicates that the peptide might be compartmentalised in the cell [26]. A dimer structure also exists in halocidin from Halocynthia papillosa, although the peptide is a heterodimer consisting of one 18- and one 15-residue monomer linked by a disulphide bond [27]. Recently, two new AMPs, halocynthin and papillosin, have been isolated from H. papillosa [29]. Both of these AMPs are active against Gram-positive and Gram-negative bacteria [29].

Alpha-helical amphipathic peptides are very common in fish as recently reviewed by Smith and Fernandes [31]. The first fish family of AMPs to be discovered was the α-helical pardaxins. These were isolated from the skin glands of Red Sea Moses sole, Pardachirus marmoratus, on the basis of their cytotoxic, pore-forming activities [48,49]. Pardaxins were originally described as toxins with anti-predatory function but subsequently they have been found to be active against Gram-positive and Gram-negative bacteria [50].

However, most fish α-helical peptides are members of the piscidin family, which includes the pleurocidins and piscidins [31]. Pleurocidins are 25-residue peptides first isolated from the skin mucus of winter flounder, Pleuronectes americanus. [51]. They have broad-spectrum antimicrobial activities [51] and inhibit DNA, RNA and protein syntheses [41]. Piscidins are 22-residue peptides first purified from skin and gills of hybrid striped bass (M. saxatilis x M. chrysops) [52] and now known to be present in other Perciformes [53]. Also within the piscidin family are dicentracin from the European bass, Dicentrarchus labrax [54], chrysophsins from red sea bream, Chrysophrys major [55] and epinecidin from the orange-spotted grouper, Epinephelus coioides [56]. All piscidins show broad-spectrum antimicrobial activity, probably killing cells via toroidal-pore formation [38,57–59]. Lee et al. [60] determined the solution structure of piscidin-1 and established that it is the conformational flexibility at the boundary between the hydrophobic and hydrophilic regions that is the critical factor for membrane selectivity and antibacterial activity. Recently, piscidin-2 has been found to cause cell membrane damage to three fungal strains known to cause infections in humans [61]. Importantly, they are expressed in mast cells [57], granules of acidophilic phagocytes and in gill, skin, stomach and intestinal epithelia [53]. In gilthead seabream, they are stored in the granules of the phagocytes and are delivered to the phagosome following uptake of bacteria by these cells [53]. Although attractive as potential candidates for topical application use because of their activity at high salt concentrations [52], the disadvantage of piscidins in this respect is their haemolytic and cytotoxic properties [57].

The first groups of cysteine-rich AMPs to be purified from invertebrates are the tachyplesins, polyphemusins, tachystatins, big defensin and tachycitin, all from the horseshoe crabs, Tachypleus tridentatus or Limulus polyphemus, as reviewed by Iwanaga [64]. Features shared by these peptides include broad-spectrum antimicrobial activities, strong amphipathicities, the presence of β-sheets and β-turns and two or more disulphide bonds [64]. The tachyplesins are 17-residue AMPs that contain an anti-parallel β-sheet linked by a β-turn, and two disulphide bonds [21,64]. The tachyplesins bind lipopolysaccharide (LPS) [21] and target the bacterial inner membrane by altering potassium permeability [65]. Their MICs range from 0.8–12.5 μg mL⁻¹ against susceptible bacteria and fungi [21]. The polyphemusins are 18-residue peptides that share with tachyplesins both β-sheets, β-turn and two disulphide bonds [21]. Polyphemusins have antimicrobial activity spectra that are similar to the tachyplesins, with MICs ranging from 3.1–12.5 μg mL⁻¹ [21].

The tachystatins of horseshoe crabs are AMPs that bind chitin, possess 3 disulphide bridges and a 3-stranded β-sheet, stabilized by cysteine residues [66]. The tachystatins share structural similarity with ω-agatoxins (venoms isolated from the funnel web spider) that also bind chitin and have weak antifungal properties [66]. The antifungal activities of tachystatins and ω-agatoxins are mediated by their chitin-binding properties but the weaker activities of ω-agatoxins (IC₅₀ values of 0.5 and 7.8 μg mL⁻¹, respectively) probably arise from the lack of strong amphipathic conformation [66]. Another horseshoe crab AMP that binds chitin is tachycitin, a 73-residue AMP containing five disulphide bonds [67]. The structure of tachycitin includes a β-hairpin loop with a 2-stranded β-sheet, and shares some sequence similarity to the hevein domain (a domain that is characteristic of peptides which bind chitin and have antimicrobial activities [67]).

The big defensin of horseshoe crabs has 79 amino acid residues and three disulphide bonds with the disulphide motif identical to the β-defensins of bovine neutrophils [20]. The N- and C-termini of the big defensin show differential antimicrobial activities and, while the N-terminus is highly active against Gram-positive bacteria, the C-terminus is most potent against Gram-negative species [20]. The big defensin has an unusual structure, containing three α-helices, a double-stranded parallel β-sheet at the N-terminus and a four-stranded anti-parallel sheet at the C-terminus, thus creating two distinct domains [68]. The N-terminus domain has a β1-α1-α2-β2 fold and contains a hydrophobic core, while the C-terminus comprises a β3-β4-β5-α3-β6 domain that adopts a compact structure held by its disulphide bonds [68]. The differential antimicrobial activities of the N- and C-termini are thought to arise from proteolytic cleavage by microbial proteinases, since cleavage with trypsin liberates the hydrophobic N and the cationic C-terminus fragments [68]. Recently, a big defensin gene (AiBD) has been cloned from the bay scallop, Argopecten irradians [69]. The deduced amino acid sequence of this gene shares 48% identity with the horseshoe crab big defensin [69]. The arrangements and spacings of the cysteine residues are highly conserved between these two AMPs, while a recombinant AiBD protein has a similar spectrum of antimicrobial activity to the horseshoe crab peptide [69].

Among the invertebrate cysteine-rich AMPs are also the defensins, myticins, mytilins and mytimycin, purified from the haemocytes of marine mussels belonging to the genus, Mytilus (M. edulis and M. galloprovincialis). These AMPs have been subjected to the most detailed investigations of all bivalve peptides reported to date [10]. All the Mytilus spp. AMPs are amphipathic and have hydrophobic and cationic features, although there are variations in their structures and activities [10]. Whereas the M. edulis defensin has six cysteines like the arthropod defensins [70], the defensin purified from M. galloprovincialis (MGD-1) has eight cysteines arranged in four disulphide bonds [63]. MGD-1 is principally active against Gram-positive bacteria [71] and contains the cysteine-stabilized α-β motif (CSαβ; [72]). Its antimicrobial activity is dependent on the β-hairpin loop that is involved in binding, growth inhibition and bacterial membrane permeabilization [71]. In contrast to the insect defensins, neither the defensin nor mytilin B from M. galloprovincialis appear to be inducible, as transcription appears to decrease with bacterial challenge, although plasma levels of the peptide increase [73,74].

The peptides in the myticin family are structurally similar to the Mytilus spp. defensins in that they have eight cysteine residues, comprising four disulphide bonds, and are potently active against Gram-positive bacteria [75]. The mytilin peptides exhibit diverse antimicrobial spectra depending on their different isoforms. Of the five mytilin isoforms so far purified from Mytilus spp., three (B, C and D) are active against Gram-positive and Gram-negative bacteria, one (G1) is active only against Gram-positive strains, while two (B and D) are also antifungal [74]. Recently the full structure of synthetic mytilin was found to share similarities with MGD-1 as both peptides contain the CSαβ motif, although the N-terminus section of mytilin is shorter and the β-hairpin more extensive in the mytilin peptide [76]. The antifungal peptide mytimycin from M. edulis has received less attention compared with the other Mytilus spp. AMPs, but is ~6.5 kDa and is thought to contain 12 cysteines [70]. In comparison with other marine invertebrates, Mytilus spp. AMPs have been the best studied with respect to their localization in cells and tissues, their expression under different conditions [77] and during their life-history [78], their differential involvement in antimicrobial defence [79] and the distribution of different isoforms in individual animals [80].

Defensins have also been purified from the gills of the American oyster, Crassostrea virginica [81] and identified at the molecular level from the mantle tissue and haemocytes of the Pacific oyster, Crassostrea gigas [32,33]. Like the Mytilus spp. defensins, the defensins from C. gigas have eight cysteines, the CSαβ motif and are principally active against Gram-positive bacteria [32]. The 38-residue defensin from C. virginica has high sequence homology (62%–73%) with the arthropod defensins, has six cysteines and is active against both Gram-positive and Gram-negative bacteria [81]. A few AMPs have been reported from clams, scallops and abalone, but as most of these have arisen from molecular methodologies relatively little is known about the features or activities of either native or recombinant peptides [10].

The two main families of cysteine-rich AMPs from crustaceans are the crustins [14] and penaeidins [17]. Carcinin, the first crustin to be discovered, was purified from the shore crab, Carcinus maenas [82], however it was not designated a crustin until much later [83]. Crustins have been found in every decapod crustacean studied, with gene sequences similar to crustins also present in the amphipod, Gammarus pulex, the brine shrimp, Artemia salina, and the copepod, Calanus finmarchius as discussed by Smith et al. [14]. Although first identified by their antimicrobial activities, the crustins are not especially potent nor do they exhibit broad-spectra antimicrobial activities, as they tend to affect mainly Gram-positive bacteria. Smith et al. [14] define crustins as cationic, cysteine-rich antibacterial polypeptides, ~7–14 kDa, containing one whey four-disulphide core domain (WFDC; also known as a WAP domain), a conserved structure of eight cysteines forming a 4-disulphide core at the C-terminus. In addition, Smith et al. [14] propose that three types of crustin (I, II, III) exist, each distinguished by the domain structure between the signal sequences and the C-terminus domain containing the WFDC. All three types possess a signal sequence at the N-terminus and the WFDC domain at the C-terminus [14]. The WFDC domain is tightly constrained by three disulphide bonds and has a small α-helix that probably accounts for the antibacterial effect of the crustins. As well as the WFDC domain, Type I crustins also possess a cysteine-rich domain that contains six cysteines [14]. Type II crustins have yet another domain, rich in glycines between the signal sequence and the six cysteine-region [14]. Type III crustins have neither the glycine- nor cysteine-rich domains [14]. It has been suggested that the crustins could have other roles in the normal physiology of the animals, as carcinin expression can change with varying environmental factors and with the moult cycle of the crab, yet shows little change in expression when subject to bacterial challenge [14].

The penaeidins, first identified from the shrimp Litopenaeus vannamei, [84] have now been isolated from a number of shrimp species [17]. Their distribution appears to be confined to shrimp unlike the crustins, which are present in shrimp as well as all other decapods [14]. Penaeidins are 5–6 kDa and, in addition to the signal sequence, they comprise two domains: a proline-arginine rich N-terminus and a cysteine-rich (typically six cysteine residues) C-terminus. Of the two domains, the cysteine-rich one is more compact and includes an α-helix, stabilized by three disulphide bonds. Five families of penaeidins have been classified to date, with their spectra of activities predominantly against Gram-positive bacteria and fungi [84]. Penaeidins have chitin-binding activity, which is also attributed to the C-terminus region, but the presence of a specific domain, such as a hevein domain, has not been confirmed [85].

A few cysteine-rich peptides have been purified from other invertebrate groups, e.g., aurelin from the jellyfish, Aurelia aurita [6]. Aurelin contains six cysteines forming three disulphide bridges and comprises a signal peptide, anionic propiece and a mature cationic part, thus resembling certain structural features of the defensins [6]. Unlike the defensins the distribution of cysteine residues is more reminiscent of the potassium-blocking channels of sea anemone toxins, although such a property in aurelin has yet to be investigated [6]. Aurelin is active against Gram-positive and Gram-negative bacteria, with MICs ranging from 7–22 μg mL⁻¹ [6].

The first AMPs to be isolated from any echinoderm are the strongylocins from the sea urchin, Strongylocentrotus droebachiensis [24]. These are cationic cysteine-rich peptides (5.6 and 5.8 kDa) that contain six cysteines likely to form three disulphide bonds, although the cysteine arrangement patterns differ from those observed in other AMPs with six cysteines [24]. Strongylocins exert antimicrobial activities against both Gram-positive and Gram-negative bacteria (IC₅₀ ranging from 1.3–5 μM) and they contain bromotryptophan; a feature they share with some other AMPs from marine sources [24] (e.g., hedistin [8]).

Amongst the cysteine-rich AMPs in teleost fish are three families: cathelicidins, defensins and LEAPs [31]. By screening cDNA libraries, putative cathelicidins have been found from rainbow trout, Oncorhynchus mykiss [86] and an EST database of Atlantic salmon, Salmo salar [87]. Using molecular methodology, further cathelicidins have been identified for Arctic char, Salvelinus alpines, Atlantic cod, Gadus morhua, and brook trout, Salvelinus fontinalis [88]. Moreover, cathelicidin genes have been reported for jawless fish, namely the Atlantic hagfish, Myxine glutinosa [89]. The structures of teleost cathelicidins have some features in common with mammalian cathelicidins, e.g., the presence of four cysteines at the C-terminus regions forming two disulfide bonds [87]. However, in general, the signal peptides of teleost cathelicidins tend to have fewer amino acid residues but a longer cathelin-like domain than mammals [87]. Little information is available with respect to the native peptides but synthetic rainbow trout cathelicidins are active against Gram-positive and Gram-negative bacteria [87].

Defensins from teleost fish, as with cathelicidins, have been identified by molecular methodologies rather than purification of the native peptides. Zhou et al. [90] used EST and complete genome data to identify defensins from zebrafish and pufferfish that resemble the β-defensins of birds and mammals. The fish defensins contain six conserved cysteines in the region of the mature peptide and three β-strands, although one difference to the avian and mammalian defensins is the presence of an extra helix in one of the zebrafish peptides [90]. Falco et al. [91], using a recombinant protein based on rainbow trout defensin, found it to be antiviral against viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most troublesome diseases in fish aquaculture. More recent studies have cloned three novel β-defensins from rainbow trout, all of which appear to be constitutively expressed but increase in expression during bacterial and simulated viral challenges [92]. Similarly, a β-defensin-like gene from the olive flounder, Paralichtyus olivaceus, has been identified, which is expressed in larval fish just one day after hatching, although the expression declines between 1–35 days post-hatching [93]. Moreover, β-defensin expression in juvenile fish is induced under conditions of bacterial challenge and the recombinant peptide suppresses the growth of E. coli [93].

The last major group of cysteine-rich AMPs from fish is the LEAPs (liver-expressed antimicrobial peptides) [31], the acronym reflecting the original identification of the peptide family in the human liver [94]. Peptides belonging to the LEAP family include hepcidins from several species (e.g., winter flounder, turbot and red sea bream), Sal-1 and Sal-2 from Atlantic salmon, JF-1 and JF-2 from Japenese flounder, and LEAP-2 from catfish and trout (reviewed by Smith and Fernandes [31]). The first fish LEAP to be identified, the hepcidin from the gills of striped bass [95], has a similar structure to human hepcidin and consists of two anti-parallel β-sheets and eight cysteines forming four disulphide bonds [96]. However, two LEAPs have since been found that only possess two disulphide bonds [31]. Their activity spectra have been determined mainly using synthetic peptides, which show activity against Gram-negative bacteria and fungi [96]. Under conditions of bacterial challenge, the expression of hybrid bass hepcidin is detectable in most tissues, but is highly up-regulated in the liver (>4,000 fold) [95]. Some fish LEAPs, e.g., those from catfish and turbot, are expressed very early in the life cycle in comparison with other fish AMPs and their involvement in iron regulation has also been suggested [31].

Histones are proteins present in the nuclei of all eukaryotic organisms. They are conspicuous proteins of chromatin and are responsible for the packaging of DNA, essentially serving to wind up the long DNA strands in a spool-like manner. There are several types of histone of which H2A, H2B, H3 and H4 are the core histones that form the nucleosome, and H1 and H5 are the linker histones. That histones have potent antimicrobial properties has been known for over 50 years [99] but at that time there was no theoretical concept as to how they might interact with bacteria for the host’s benefit, so little attention was paid to this discovery. By the late 1990s, histones were reported to contribute to the antibacterial activity of wound blister fluid [100]. Then reports began to emerge that histones account for a large proportion of the antibacterial activity of skin exudates from amphibians [101] and teleost fish [102–105]. Noga et al. [106] also found that histones are active against fish-parasitic dinoflagellates. Histones have now been reported to be present in the skin mucus of several fish taxa, including Salmoniformes, Siluriformes and Pleuronectiformes [31] (Table 3).

Amongst the core histones, H2A is a potent antibacterial agent. It is a 13.6 kDa protein able to kill Gram-positive bacteria at sub-micromolar (<0.4 μM) concentrations within 30 minutes in vitro [105]. It also has some weak activity against the yeast, Saccharomyces cerevisiae, but it is not haemolytic to trout erythrocytes at antimicrobial concentrations [105]. It does not appear to form stable ion channels in the bacterial cell membrane but reconstituting pure H2A in a planar lipid bilayer does disturb the membrane [105]. Interestingly, it is not only the intact H2A protein that has antimicrobial effects but also fragments generated from the N-terminus by proteolytic cleavage (Table 3). Such fragments include parasin-1 from the catfish, Parasilurus asetu, [101], hipposin from Atlantic halibut Hippoglossus hippoglossus [109], buforins from toads [114], and abhisin from abalone [110]. H2A or fragments derived from it have also been recorded for scallop, Chlamys farreri [115] and shrimp, Litopenaeus vannamei [108] (Table 3). The liberation of parasin-1 from H2A in fish mucus is mediated through the enzyme cathepsin D [116] with a second enzyme, matrix metalloproteinase 2, involved in the regulation of this process [117].

Core histone H2B also has antimicrobial activity, which was first noted for mouse macrophages [118] and subsequently reported for channel catfish skin exudates [102], epidermal secretions from Schlegel’s Green tree frog [119], surface mucus from Atlantic cod [111] and shrimp haemocytes [108] (Table 3). Histone H2B is active against the fish pathogens, Aeromonas hydrophilia and Saprolegnia [102]. Histone H4 is another microbicidal core histone, having been found to be one of the active factors in human sebocyte secretions [120]. H4 from shrimp haemocytes has antibacterial properties [108] but reports for its antimicrobial role in other marine or aquatic organisms are scant. With H3, synthetic H3–like peptides are antibacterial [121] and H3 is present in mucus extruded from the hagfish Myxine glutinous [122].

The linker histones also have anti-infective properties (Table 3). H1 exhibits antimicrobial properties and has been isolated from several species, including humans [123], mice [118], fish [104,107,124] and shrimp [108]. H1, when isolated from Coho salmon, is active against E. coli with a MIC of 31 μg mL⁻¹ [104]. A 26-residue N-terminus fragment of H1 from Coho salmon is also active against various fish pathogens, including Aeromonas salmonicida, Listonella anguillarum and Salmonella enteritica [103]. In winter flounder, the expression of this protein is up-regulated following immune stimulation, which coincides with an increase in the antibacterial activities of serum and mucus [103], indicative of its role in systemic as well as mucosal response to non-self challenge. Significantly, the C-terminus from H1 (from O. mykiss) has been shown to generate a 7.2 kDa fragment, termed oncorhyncin II, that has very high potency (~10 times greater than cecropin P1) against both Gram-positive and Gram-negative bacteria, probably by destabilizing the bacterial membrane, although not necessarily by pore formation [107]. Therefore, H1 is strongly antibacterial not only as the complete molecule but through fragments at both the N and C-termini (Table 3).

Histones are highly conserved alkaline and water soluble proteins showing remarkable similarity across divergent phyla. For example, antisera to human histones are known to cross react with histones from several invertebrates [125]. It is therefore highly likely that histones from most, if not all eukaryotic species, will show similar potent antimicrobial activity as those described above, although the extent and modes of histone participation in host defence is, as yet, far from clear. Their possession of amphipathic secondary structures and microbicidal properties might be merely incidental and have no survival value. Hirsch’s finding in 1958 [99] of the antimicrobial properties of histones was disregarded as not physiologically relevant for many years but the presence of these peptides in skin secretions of fish is less surprising given that fish skin is living and not keratinized. The epithelial surface is constantly at risk of abrasion and sloughing, so damage to the cells from such minor injuries might permit histones and other intracellular antibacterial proteins, along with phospholipid-derived free fatty acids, to become exposed to epibionts or potential invaders from the surrounding water. Enzymes, such as cathepsin D, also expressed in fish epidermal mucosa, are now known to aid the release of histone fragments [116]. However, this does not explain how histones or other intracellular antibacterial factors might interact with infectious agents within the body tissues of any animal.

Apart from the core and linker histones, research on rainbow trout has established that other intracellular proteins have potent antibacterial activities against infectious agents. One from fish is a 6–7 kDa N-terminus fragment of a high mobility non-histone chromosome protein, H6, which has very powerful activity against a range of bacteria (MICs 0.06–0.12 μM) [112]. This fragment, designated as oncorhyncin III, is salt sensitive, non-haemolytic and able to destabilize planar lipid membranes [112]. Another intracellular protein found to have antibacterial effects is a 6.6 kDa fragment derived from the 40S ribosomal protein, called S30 [113] (Table 3) from skin secretions of O. mykiss. S30 inhibits the growth of Gram-positive and Gram-negative bacteria but has strongest microbicidal activity against the Gram-positive species [113]. Three ribosomal-derived proteins and peptides, namely L40, L36A and L35, (6.4, 12.3 and 14. 2 kDa, respectively), have also been isolated from the epidermal mucus of Atlantic cod, Gadus morhua [111] (Table 3). The remarkably strong activity of core and linker histones, as well as ribosome-derived proteins, raises questions about the contribution of their anti-infective properties to defence, as they would normally not be exposed to invasive bacteria, even intracellular ones.

The cell membranes of eukaryotic organisms can also be an important source of several antimicrobial compounds, with free fatty acids (FFAs) particularly prominent. Fatty acids, especially unsaturated varieties, are rarely found in their free form inside living cells [126,127] but more usually they are bound to other groups, such as phosphates, sugars or glycerol, forming lipids. Phospholipids are the major structural components of cell membranes, while galactolipids are located in the chloroplasts and triglyercides may form cellular energy reserves as lipid stores. Fatty acids can be released from cell membranes as FFAs upon cell or tissue damage caused typically by a consumer or a pathogen [126–130].

There are numerous FFAs with antimicrobial effects and their spectra of action and potencies are influenced by the degree of saturation, length of carbon chain and the orientation of the double bonds [131] (Table 4). Briefly, amongst the saturated FFAs, capric acid (C10:0) and lauric acid (C12:0) tend to be the most active [132–134], while amongst the monounsaturated FFAs, myristoleic (C14:1) and palmitoleic acid (C16:1) often are the most potent [132,133] (Table 4). Monounsaturated FFAs with less than 14 or more than 16 carbon atoms in the carbon chain tend to have rather less activity [132,133] (Table 4). Many polyunsaturated FFAs are potently antibacterial, particularly those with 18 and 20 carbons in their carbon chain [132]. Interestingly, a direct relationship can exist between the number of double bonds in the carbon chain and the antibacterial activity of the FFA [132,135,136] (Table 4). In general FFAs with cis-orientated carbon-carbon double bonds have greater antimicrobial activities compared with those FFAs containing trans-orientated bonds [132,137] (Table 4).

The microbicidal activities of membrane-derived FFAs have been extensively studied and these seem to be important defence effectors, especially in the macro- and micro-algae [126,128–131]. Micro-algae, even those species with partial or complete cell armouring, such as diatoms, can lose structural integrity through mechanical damage by consumers, osmotic shock, water turbulence and viral- or bacterial-induced lysis. Such damage may result in the release of high concentrations of FFAs, particularly from the phospholipids of the cell membrane and the galactolipids of the chloroplasts, into the vicinity of the damaged cell [127,138,139]. The liberation of FFAs is immediate and is carried out by a family of hydrolytic enzymes called lipases [127,140]. During pathogen-induced lysis the bioactive FFAs are thus brought into close contact with any adjacent prokaryotes, whether they are the same pathogen that may have initiated the damage in the first instance, or are other heterotrophic opportunists. Killing of these microbes by the released FFA will not, of course, save the damaged micro-alga but could help protect its neighbours and would be beneficial, in evolutionary terms, to the population if these surrounding cells are clonal or close genetic relatives. In this way, pathogen transmission within a micro-algal population can be brought under some control.

The production of FFAs is a multi-faceted defence strategy in the micro-algae, as these compounds are toxic to many threats to host survival, including viruses, protozoans and consumers [126,141]. Further, this defence strategy can be considered metabolically inexpensive, as the FFAs come from vital structures within the cell and the lipases involved may pre-exist to serve alternative essential functions in living cells [142,143]. While micro-alga-pathogen interactions are not well characterised there is a growing literature in this field [144–147] but future experimentation must confirm the precise role for FFAs against recognised micro-algal pathogens. Pathogen-damaged tissues of macro-algae produce FFAs in a similar manner to the micro-algae and these not only kill the pathogen and prevent its spread through the host but the FFAs can also act as signals (or precursors of signalling molecules) that trigger downstream systemic defence responses [128–130].

The marine diatom Phaeodactylum tricornutum is a popular model for investigations of micro-algal physiology and it is a good example of a micro-alga that forms antibacterial FFAs through lipase action after mechanical disruption of the cells [148]. The main ones are medium- and long-chain unsaturated varieties, particularly eicosapaentaenoic acid (C20:5 n-3) [149], hexadecatrienoic acid (C16:3 n-4) and palmitoleic acid (C16:1 n-7) [150]. These unsaturated FFAs are typical of those isolated from extracts in similar antibacterial bioassay-guided fractionation studies of other macro- and micro-algal species [151,152]. Eicosapaentaenoic acid (EPA) has strong activity against a wide range of Gram-positive and Gram-negative marine and non-marine bacteria in vitro, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) [149] (Table 5).

Fish and shellfish pathogens, such as Lactococcus garviae, harmful Vibrios and L. anguillarum are also killed by EPA although it does not appear to affect fungi [149,153] (Table 5). Similarly, hexadecatrienoic acid (HTA) isolated from P. tricornutum displays activity against the Gram-positive pathogen, S. aureus [150]. Palmitoleic acid (PA) is active against various non-marine Gram-positive human pathogens at micromolar concentrations and begins to kill bacteria upon immediate exposure [150]. Like EPA, HTA appears to have little or no activity against fungi [150], although there are reports of antifungal activity attributable to PA [154]. Interestingly, higher levels of these and other bioactive FFAs are present in the fusiform morphotype of P. tricornutum (the morphotype that tends to dominate in the plankton) compared with the oval morphs of this micro-alga, which tend to occur on surfaces [155]. It is likely that this is a functional adaptation to prevailing conditions but shows that yields of antibacterial FFAs, both naturally in the sea and experimentally in the laboratory, can be influenced by growth and environmental conditions. MICs for FFAs against susceptible bacteria are typically 100 μM and greater [133,156–158] but, in certain cases, they can be as much as one order of magnitude more potent [133,135,159]. However, whilst FFAs may not be as potent as AMPs by direct comparison, their fast accumulation and thus potentially greater local concentrations mean that they can attain the necessary concentrations to exert their antimicrobial activities.

FFAs can be bactericidal (kill ≥99.9% of original inoculum) or can reversibly inhibit bacterial growth without complete killing (bacteriostatis) [160], although the mechanisms by which they act have yet to be fully characterised. There are several ways that they are thought to attack bacterial cells but, of these, the cell membrane is probably the prime target [131]. Likely cell processes targeted by FFAs include interference with cellular energy production by disrupting the electron transport chain and oxidative phosphorylation, inhibition of enzyme activity, impairment of nutrient uptake, generation of toxic peroxidation and auto-oxidation degradation products or direct membrane disruption causing bacterial cell lysis [131,161]. The exact process responsible is likely to depend on the bacterial strain, the FFA concerned and its concentration. It is also probable that FFAs work simultaneously on multiple targets within the bacterial cell, thus reducing the likelihood of inducible bacterial resistance.

In many species of macro- and micro-algae, the FFAs released by lipases from the phospholipids and galactolipids of damaged cells are very rapidly transformed into other compounds [127,130,138,139,162]. The usual process is for the FFA to be oxygenated initially by lipoxygenase enzymes to give intermediary hydroperoxides [163,164]. Further enzymes convert the hydroperoxides into oxylipins, of which there is a great variety, including unsaturated aldehydes and hydroxyl-, keto- and epoxyhydroxy fatty acid derivatives [165–168]. The exact repertoire of FFA-derived oxylipins depends on the algal species (and sometimes the strain) and the particular suite of enzymes that they express [169].

Importantly, the oxylipins produced may have appreciably more or, in some cases, less antibacterial activity than the FFA from which it was derived [136,170]. Amongst the micro-algal-derived oxylipins, it is the antibacterial activities of the polyunsaturated aldehydes (PUAs) that have attracted most recent attention. These PUAs are generated by diatoms including Skeletonema costatum and Thalassiosira rotula [169]. Diatom-derived PUAs are produced from C16, C20 and C22 polyunsaturated FFAs, particularly HTA, hexadecatetraenoic acid (C16:4 n-1) and EPA [138,139,162–164]. These FFAs are first oxygenated by a lipoxygenase and then hydroperoxide lyases act on the hydroperoxide intermediates to give PUAs, including heptadienal (C7:2 n-3), octadienal (C8:2 n-4), octatrienal (C8:3 n-1) and decadienal (C10:2 n-3) [138,139,162–164]. One typical, well-studied and highly antibacterial PUA is decadienal (DD), which is probably derived from the polyunsaturated fatty acid, arachidonic acid (C20:4 n-3) [127,141]. DD exhibits strong activity against important Gram-positive and Gram-negative human pathogens, such as MRSA and Haemophilus influenzae with MIC values of 7.8 and 1.9 μg mL⁻¹, respectively [171]. DD at micromolar concentrations also detrimentally affects the growth of diverse Gram-positive and Gram-negative marine bacteria [172,173] (Table 5). Indeed, DD is also highly antagonistic towards numerous non-marine bacterial species (Table 5) and there is also evidence for activity against fungi [172]. Little work has been performed to characterise the antibacterial activities of PUAs and further studies in this field are warranted. Importantly, it is necessary to discriminate between bactericidal and bacteristatic activities. Such studies would begin to elucidate their specific mechanism(s) of antibacterial action.

The antimicrobial properties of mammalian haemoglobin have been known for a long time [206] and the activities are now attributed to peptides derived from the α- and β-subunits within the haemoglobin tetramer [207,208]. With fish, Fernandes and Smith [174] have reported the partial purification by RP-HPLC of two cationic proteinaceous factors from the acid soluble erythrocyte extracts of O. mykiss (Table 6). One of these factors is active against Gram-positive and Gram-negative bacteria with MIC values of 7–14 μg mL⁻¹ and 14–28 μg mL⁻¹, respectively, while the other is active against the Gram-positive bacterium, Planococcus citreus, with a MIC of 1–2 μg mL⁻¹ [174] (Table 6). All of these values are within the sub-micromolar range and are approximately in the same order of magnitude as cecropin P1 but, as yet, the exact nature of these proteins is unknown [174]. These antimicrobial compounds could be haemoglobin-derived peptides but, as fish have nucleated erythrocytes, it is also possible that one or more of the activities might be due to histones or other intracellular structures, such as those described above. Other studies have established that two peptides derived from the β-subunit of catfish (Ictalurus punctata) haemoglobin have antibacterial activity against Gram-negative bacteria and these peptides can also kill Ichthyophthirius multifiliis, a ciliate parasite of this fish [209] (Table 6). As the transcript for at least one of these β-subunit-derived antimicrobial peptides from haemoglobin seems to be up-regulated in the skin and gill epithelium of I. punctata following I. multifiliis infection, it likely that these peptides might act in an anti-infective manner for the host [209].

Haemocyanin is another important oxygen carrying pigment in the blood of many invertebrates, especially large active ones, such as decapod crustaceans. Several studies have revealed its involvement in an anti-infective capacity (Table 6). With shrimp, intact haemocyanin can not only directly agglutinate bacterial cells [210] thereby restricting their ability to divide and spread around the body, but it also has antiviral effects [211].

Perhaps more interestingly, two antifungal peptides of 2.7 and 7.0–8.3 kDa, respectively are generated by limited proteolytic cleavage at the C-terminus of haemocyanin from the shrimp, Litopenaeus vannamei [175]. These peptides have strong activities against a range of fungi with MIC values of ~3–12 μM [175] (Table 6). They probably serve to kill fungi that may penetrate into the haemocoel through the carapace but their effects do not appear to be due to heavy metal or binding sites for bivalent ions [175]. In addition to these, another antibacterial peptide, astacidin-1, appears to be generated from the C-terminus of haemocyanin from the crayfish, Pacifastacus leniusculus, by proteolytic cleavage in acidic conditions [212]. This peptide is active against a range of Gram-positive and Gram-negative bacteria, including some human pathogens, at MIC values ranging from 2–20 μM [212]. The bioactivity of astacidin-1, which contains a β-sheet like many conventional AMPs, seems to reside at its N-terminus and its importance in host defence is demonstrated by its increased release into the haemolymph after crayfish have received injections of bacterial LPS [212].

An example of a non-respiratory blood pigment that has antimicrobial effects is echinochrome A from echinoderms, particularly sea urchins (Table 6). Echinochrome is an orange-red coloured naphthoquinone (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone) that has iron chelating and free radical scavenging properties [213]. Echinochrome A is abundant in the red spherule coelomocytes of echinoid coelomic fluid [214]. It was found to have bactericidal properties by Service and Wardlaw [176] working with the common European sea urchin, Echinus esculentus. In this species it is present in the cells at 3–60 μg mL⁻¹ and, importantly, it is active against a range of Gram-positive and Gram-negative bacteria at 50 μg mL⁻¹ [176] (Table 6). It is highly likely to owe its antibacterial effects to iron-chelation and, as it is also present in urchin eggs and larvae, it may assist in preventing microbial colonization after spawning and fertilization.

Melanin is a pigment that occurs in many organisms: animals, plants, fungi and bacteria. Indeed, for many marine protostome invertebrates, particularly crustaceans and molluscs, and to a lesser extent, in annelids and ascidians, melanisation is a conspicuous immune response to infection. In these animals, wounds and abrasions to the external body surface rapidly become melanised, while internally melanin deposition accompanies the formation of haemocyte capsules around fungi, parasites or bacteria that have gained access to the haemocoel or coelomic cavity. The occurrence of melanin at sites of injury or infection in so many taxa has led to the general conception that melanin, and/or its quinone precursors, have anti-infective properties (Table 6) and a number of papers have appeared offering experimental data to support this [177–180]. However, it has also been argued that melanin is not a significant anti-infective, particularly as some fungi and pathogenic bacteria defend themselves against attack by the host immune system by utilizing melanin, either synthesized by themselves or produced by the host [215]. Certainly melanin has anti-oxidant properties, particularly through mopping up H₂O₂, so it may be exploited by some pathogens for protection against the effects of reactive oxygen intermediates generated by the host phagocytes. However other invaders, such as fungal hyphae, are essentially sequestered by this insoluble polymer into the haemocyte capsule matrix and then asphyxiated.

What may be important in relation to any anti-septic benefits of melanisation, at least in some protostome invertebrates, is the enzymatic oxidation of phenolic molecules to form the quinone precursors of melanin. The enzyme responsible is phenoloxidase (PO) and the activation of PO from its inactive precursor, proPO, is known to be a key defence response to microbial infection in many marine and non-marine invertebrates. Certainly, purified phenoloxidase from blood cells of the solitary ascidian, Halocynthia roretzi, has antibacterial activity in the presence of -(3,4-dihydroxy)-phenylalanine (L-dopa) [182] and indirect evidence has been provided that PO from the cephalochordate, Amphioxus belcheri, has antibacterial effects [183] (Table 6). In arthropods, phenoloxidase-catalyzed reactions have been found to produce antimicrobial reactive intermediates from natural substrates [216] and in shrimp silencing of the gene encoding prophenoloxidase by RNA interference results in a significant increase in bacterial load in the haemolymph [217]. Importantly, too, work on crustaceans has shown that proteolytic cleavage of haemocyanin, not only generates an antibacterial peptide from the C-terminus [212] but also produces PO from the N-terminus of subunit 2 [218]. Thus there is a clear link between haemocyanin, melanization reactions and microbial killing in invertebrates.

Not all pigments that show anti-infective effects are present in blood or body fluids circulating freely through the body. Indeed, the ink produced by some cepalopods and opistobranch gastropods upon attack or threat as defensive, anti-predator shields has been found to possess a number of factors with antibacterial, antifungal and/or cytotoxic activities [184,219,220]. At least one such pigment in the ink of octopus is melanin [181] but in gastropods, especially those belonging to the genera Aplysia and Dolabella, other antimicrobial factors occur (Table 6). One is a 60 kDa glycoprotein, termed aplysianin P [184,185]. This is bacteriostatic against various Gram-positive and Gram-negative bacteria at 0.2–5.8 μg mL⁻¹ and seems to function by completely inhibiting the syntheses of bacterial DNA and RNA; a property that also lends it cytotoxic activity against eukaryotic tumour cells [184]. Related aplysianins are present in the albumin gland (aplysianin A) and eggs (aplysianin E) of A. kurodai [221,222]. The latter must certainly help the eggs from being colonized by bacterial and fungal epibionts. All of these aplysianins possess l-amino acid oxidase (LAAO) activity placing them within the family of LAAO enzymes. As the name suggests these deaminate l-amino acids in an oxidative manner to yield various microbicidal molecules, including H₂O₂, NH⁺ ions, carboxyl acids and α-keto acids [186,187,223]. A wide variety of LAAOs occur in marine and other organisms and most have broad-spectrum anti-infective properties [186]. One, designated Sebastes schlegeli antibacterial protein, is present in skin secretions of the rockfish, Sebastes schlegeli, and is active against the Gram-negative fish pathogens A. hydrophila, A. salmonicida and Photobacterium damselae ssp. piscicida [187] (Table 6). It has a yellow colouration and is derived from a 120 kDa homodimeric glycoprotein [187] (Table 6). Different LAAOs have different substrate specificity but the generation of the bactericidal products is usually very rapid [224]. One LAAO homolog of the aplysianins in sea hare ink is escapin, a 60 kDa monomer that has high oxidase activity with either l-lysine or l-arginine, and seems to kill actively growing bacteria by a variety of mechanisms, including hydrogen peroxide release [224]. Escapin is a highly stable compound that preferentially kills Gram-negative bacteria at MIC of ~0.25–0.62 μM mL⁻¹, although it also prevents the growth of some Gram-positives, including S. aureus and has mild activity against certain fungi [224].

With micro-algae, several photosynthetic pigments or derivatives have been isolated and found to have microbicidal effects (Table 6). Jorgensen [188] reported the isolation of an antibacterial derivative of chlorophyll from extracts prepared from three different micro-algae (Chlamydomonas reinhardi, Chlorella vulgaris, Scendesmus quadricauda), which is active against the Gram-positive bacterium, Bacillus subtilis. The same compound, thought to be a photo-oxidation product of the chlorophyll derivative chlorophyllide a, was later isolated by Hansen [189] from acetone extracts of the chrysophyte, Ochromonas malhamensis. This antibacterial agent is active against Gram-positive and Gram-negative bacteria. In the same study, two further pigment derivatives with antibacterial activity were isolated but these could not be identified conclusively [189]. Finally, Bruce et al. [190] also reported the isolation of two chlorophyll a derivatives, which are active against Gram-positive and Gram-negative marine bacteria.

Importantly, too, little effort has been exerted into exploring the use of anti-infectives from marine eukaryotes in combination therapies with traditional antibiotics. Certainly synergy between different antimicrobials produced within the same organism must occur in nature, and there are a few sporadic reports of synergism occurring between co-expressed proteins in some fish or invertebrates. For example, with invertebrates, Gueguen et al. [97] used an in vitro checkerboard assay to study synergy between recombinant defensin and synthetic fragments of a 61-residue proline-rich AMP from the oyster, C. gigas. The combination of these agents produced around a threefold increase in the killing of E. coli [97]. Synergism between tachycitin and big defensin, two conventional antimicrobial peptides from amoebocytes of horseshoe crab, Tachypleus tridentatus, has also been reported [264]. As little as 0.9 μg mL⁻¹ tachycitin reduces the IC₅₀ of big defensin from 0.8 μg mL⁻¹ to 0.015 μg mL⁻¹ [264].

Lysozyme is another important anti-infective in a wide range of coelomate animals that can synergize with other anti-infectives. It occurs very widely across the animal kingdom, often in multiple forms and has the effect of breaking open the peptidoglycan cell wall of Gram-positive bacteria, thus allowing AMPs to enter the cell and permeabilize the inner leaflet. Accordingly it can serve both as a direct effector and as synergiser of AMP activity. Notably, teleost fish possess multiple types of lysozyme-like muramidases, especially in skin secretions. These include not only the conventional cationic lysozymes but also an unusual anionic type of muramidase, which has yet to be fully characterised [265]. Concha et al. [197] has also reported that lysozyme enhances the antimicrobial ability of apoA-1 from carp, and a further example of synergism between different antimicrobial proteins is given by the work of Patrzykat et al. [103]. These authors combined native pleurocidin with lysozyme, HSDF-1 or HSDF-2 (two different fragments of histone H1 from Coho salmon). These combinations produced highly synergistic improvements in inhibiting the growth of the fish pathogens, Aeromonas salmonicida and L. anguillarum [103] and shows that histones also have potential to be deployed as synergists for other antibiotic substances. Histones are known to disrupt lipid bilayers but do not seem to form pores in bacteria and do not have haemolytic effects [105,175] so could be well tolerated by patients receiving such treatments.

Oxylipins and FFAs, too, might find application in combination therapies, as synergists for non-lipid anti-infectives, including some of those described above. Unfortunately few studies have investigated the potential of oxylipins, despite their known activities against important pathogens [172] (Table 5). By contrast FFAs are already on the agenda for exploitation as bio-pharmaceuticals alone or in synergy with anti-infectives for use in medicine [159,266,267]. The great advantage of FFAs lies in their broad-spectra of activity, high potency, relative safety and the lack of inducible resistant phenotypes [134,268–271]. Those from micro-algal extracts should be of high quality, cheap and easy to produce on a commercial scale and have already shown particular promise in formulations to prevent the colonisation of human skin by opportunistic pathogens, such as MRSA [272]. Fatty acid-enriched gels have been produced that may be useful in preventing the spread of sexual transmitted bacterial and viral pathogens [273], while the palmitoleic acid isomer, C16:1n-10, has been found to be effective at reducing systemic staphylococcal burden in mice and therefore might prove valuable for the treatment of serious systemic infections of humans by S. aureus and MRSA [274].

A final word might be said about the possibility of using knowledge obtained about the structures and gene organisation of marine-derived anti-infectives to design chimeric compounds that effectively combine synergistic or additive motifs into a single molecule or drug. Some recent work on conventional AMPs from crustaceans has revealed that nature has already created some multi-domain AMPs, each domain of which being known from other research to have independent, potent antimicrobial properties. Good examples are afforded by hyastatin from the spider crab, Hyas arenaeus [16] and penaeidins from shrimp [17]. The penaeidin domains comprise a proline-arginine-rich region at the N-terminus and a cysteine-rich one at the C-terminus [275] (Figure 1). The proline-rich region is unstructured and forms a long tail, in contrast to the highly ordered conformation of the C-terminus, which is tightly coiled with an α-helix and three disulphide bonds [277]. The chitin-binding activity of the C-terminus is likely to contribute to the antifungal properties of the penaeidins, and may also allow the peptide to become anchored to the carapace during wound healing or at the moult [276]. The N-terminus proline-arginine-rich domain, however, seems to account for the antibacterial properties of the whole molecule [278].

The second example from crustaceans, hyastatin, is made up of three domains and, like penaeidins and many conventional AMPs, is liberated from its inactive precursor by proteolytic cleavage of the N-terminus signal sequence [16] (Figure 1).

The N-terminus of the mature protein has a glycine-rich (LGGG/IGGG) domain and a C-terminus cysteine-rich domain with six cysteine residues that probably form an α-helical configuration with three disulphide bonds, while in between the termini is a short proline-arginine-rich region [16] (Figure 1). The glycine-rich domain has a striking resemblance to the glycine-rich domain of shrimp Type II crustins while the C-terminus and central proline-arginine domains are similar to the proline-arginine- and cysteine-rich domains of penaeidins [16]. Thus hyastatin appears to be chimeric, comprising domains that characterise and account for the functionality of other, quite distinct anti-infective proteins in separate crustacean groups. Interestingly, hyastatin seems to owe its antibacterial properties to the cysteine-rich C-terminus domain, like crustins [14], because a recombinant protein lacking the cysteine-rich domain has no bactericidal activity [16]. However, as mentioned earlier when considering conventional, amino acid enriched AMPs, the chitin-binding properties may indicate that the peptide is multifunctional. Nature thus seems to ‘mix and match’ useful domains to create AMPs that could have dual mechanisms to destroy their targets. The possibility remains that synthetic anti-infectives might be designed and engineered that are also chimeric but use structures known to have particular efficacy against certain types of pathogen to maximize killing and reduce the risk of resistance.