Tissue localization of piscidin host-defense peptides during striped bass (Morone saxatilis) development.

Infectious diseases are a major cause of larval mortality in finfish aquaculture. Understanding ontogeny of the fish immune system and thus developmental timing of protective immune tissues and cells, may help to decrease serious losses of larval fishes when they are particularly vulnerable to infection. One component of the innate immune system of fishes is the host-defense peptides, which include the piscidins. Piscidins are small, amphipathic, α-helical peptides with a broad-spectrum of action against viral, bacterial, fungal, and protozoan pathogens. We describe for the first time the cellular and tissue localization of three different piscidins (1, 3, and 4) during striped bass (Morone saxatilis) larval ontogeny using immunofluorescent histochemistry. From 16 days post hatch to 12 months of age, piscidin staining was observed in cells of the epithelial tissues of gill, digestive tract, and skin, mainly in mast cells. Staining was also seen in presumptive hematopoietic cells in the head kidney. The three piscidins showed variable cellular and tissue staining patterns, possibly relating to differences in tissue susceptibility or pathogen specificity. This furthers our observation that the piscidins are not a monolithic family of antimicrobials, but that different AMPs have different (more specialized) functions. Furthermore, no immunofluorescent staining of piscidins was observed in post-vitellogenic oocytes, embryos, or larvae from hatch to 14 days post hatch, indicating that this critical component of the innate immune system is inactive in pre-hatch and young larval striped bass.

A Diverse Family of Host-Defense Peptides (Piscidins) Exhibit Specialized Anti-Bacterial and Anti-Protozoal Activities in Fishes.

Conventional antibiotics and other chemical-based drugs are currently one of the most common methods used to control disease-related mortality in animal agriculture. Use of the innate immune system to decrease disease related mortalities is a novel alternative to conventional drugs. One component of the innate immune system is the host-defense peptides, also known as antimicrobial peptides. Host-defense peptides are typically small, amphipathic, α-helical peptides with a broad-spectrum of action against viral, bacterial, fungal, and/or protozoal pathogens. Piscidins are host-defense peptides first discovered in the hybrid striped bass (white bass, Morone chrysops, x striped bass, M. saxatilis). In this paper we identify four new piscidin isoforms in the hybrid striped bass and describe their tissue distributions. We also determine the progenitor species of origin of each piscidin (orthology) and propose a revised nomenclature for this newly described piscidin family based on a three class system. The Class I piscidins (22 amino acids in length; striped bass and white bass piscidin 1 and piscidin 3) show broad-spectrum activity against bacteria and ciliated protozoans, while the Class III piscidins (55 amino acids in length; striped bass and white bass piscidin 6 and striped bass piscidin 7) primarily show anti-protozoal activity. The Class II piscidins (44-46 amino acids in length; striped bass and white bass piscidin 4 and white bass piscidin 5) have a level of activity against bacteria and protozoans intermediate to Classes I and III. Knowledge of piscidin function and activity may help in the future development of disease-resistant lines of striped bass and white bass that could be used to produce superior hybrids for aquaculture.

The effect of Ochratoxin A on antimicrobial polypeptide expression and resistance to water mold infection in channel catfish (Ictalurus punctatus).

Mycotoxin contamination of agricultural commodities poses a serious risk to animal health, including aquaculture species. Ochratoxin A (OA) is the most immunotoxic ochratoxin, yet little is known about its effect on immune function in fish. Antimicrobial polypeptides (AMPPs) are one of the most potent, innate, host defense factors, yet very little is known about what types of chronic stressors affect their expression. Among the most prevalent and potent AMPPs in fish are histone-like proteins (HLPs). In this study, fish were fed 2, 4, or 8 mg OA/kg diet. Skin antibacterial activity and HLP-1 levels were measured on Days 0, 28 and 56. Feeding 2, 4 or 8 mg OA/kg diet resulted in significant growth depression, but higher levels (4 or 8 mg OA/kg diet) resulted in lowering feed intake (FI) and impaired feed conversion ratio. In addition, feeding 8 mg OA/kg diet increased susceptibility to experimental water mold (Saprolegnia) challenge, suggesting that OA toxicity might contribute to some saprolegnosis outbreaks. However, there were no changes in AMPP expression in any treatment group. Our data suggests that the increased disease susceptibility of channel catfish due to OA is probably due to mechanisms other than a direct effect on antimicrobial polypeptide expression.

Piscidins in the intestine of European perch, Perca fluviatilis, naturally infected with an enteric worm.

This study set out to determine how an enteric parasite, the thorny-headed worm Acanthocephalus lucii, affected the expression of antimicrobial peptides (piscidins) in its host population, the European perch (Perca fluviatilis) collected from Lake Piediluco in Central Italy. A total of 87 perch were examined; 44 (50.5%) were infected with A. lucii (1-18 worms fish(-1)). Pathological changes and immune response were assessed using histological, ultrastructural and immunohistochemical techniques. The acanthocephalans only penetrated the surficial zone of the intestinal wall and induced only slight inflammation. The main damage was destruction of the mucosal epithelium covering the villi adjacent to the parasite's attachment site, and included necrosis and degeneration. Infected intestine had numerous mast cells (MCs), often in close proximity to, and within, the capillaries, and were associated with fibroblasts of the submucosal layer. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bounded granules. Immunostaining of intestine with antibodies against the antimicrobial peptides piscidin 3 and piscidin 4 showed subpopulations of MCs that were positive. Piscidin-positive MCs were mainly observed among the epithelial cells of the intestine, but also within the submucosa. In both uninfected and parasite-infected perch, the number of MCs positive for piscidin 4 was higher than those immunoreactive with piscidin 3 (p < 0.05). For both piscidins, there was no significant difference in the number of positive MCs between parasite-infected and uninfected intestine (p > 0.05). However, uninfected fish showed higher immunostaining intensity for piscidin 3 than infected conspecifics (p < 0.05).

Mast cells in common wolffish Anarhichas lupus L.: ontogeny, distribution and association with lymphatic vessels.

The morphology, ontogeny and tissue distribution of mast cells were studied in common wolffish(Anarhichas lupus L.) at the larval, juvenile and adult life stages using light and electron-microscopy and immunohistochemistry. Fish were sampled at 1 day, 1, 2, 3, 4, 8 and 12 weeks post-hatching in addition to 6 and 9 months and 2 years and older. From 8 weeks post-hatching, mast cells in common wolffish mainly appeared as oval or rounded cells 8-15 mm in diameter with an eccentrically placed, ovoid nucleus and filled with cytoplasmic granules up to 1.2 mm in diameter. Granules were refractile and eosinophilic to slightly basophilic in H&E and stained bright red with Martius-scarlet-blue and purple with pinacyanol erythrosinate in formalin-fixed tissues. Mast cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 1 day to 4 weeks post-hatching, immature mast cell containing only a few irregularly sized cytoplasmic granules were observed by light and electron-microscopy in loose connective tissue of cranial areas. From 1 day post-hatching, these cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 12 weeks post-hatching, mast cells showed a primarily perivascular distribution and were particularly closely associated with lymphatic vessels and sinuses. Mast cells were mainly located at the peripheral border of the adventitia of arteries and veins, while they were in intimate contact with the endothelium of the lymphatic vessels. Numerous mast cells were observed in the intestine. A stratum compactum, as described in salmonids, was not observed in wolffish intestine,nor were mast cells confined to a separate layer, a stratum granulosum. Lymphatic vessels consisting of endothelium, intimal connective tissue and a poorly developed basal lamina were observed in the intestine. Scanning electron microscopy was used to compare the structure and localization of intestinal mast cells of common wolffish and rainbow trout. Scanning electron microscopy also revealed endothelial surface features and confirmed the existence of three distinctly different types of vessels in the wolffish intestine. Rainbow trout mast cell granules appeared as intact globular structures while empty vacuoles were observed in common wolffish. Mast cells were closely associated with lymphatic vessels in common wolffish, but not in rainbow trout.

The effect of adjuvant and microbial challenge on the expression of antimicrobial polypeptides in channel catfish (Ictalurus punctatus).

While antimicrobial polypeptides (AMPPs) are increasingly recognized as one of the most important components of innate immunity, there is very little information in vertebrates that documents their upregulation to levels that are microbicidal in vivo. Here we demonstrate that intraperitoneal injection of either Freund's complete adjuvant (FCA) or live Tetrahymena pyriformis (a parasitic ciliate) upregulated AMPP expression in channel catfish skin. FCA induced significant upregulation of total antibacterial activity, anti-Edwardsiella ictaluri activity (the fraction of antibacterial activity active against E. ictaluri), and HLP-1 (the major AMPP in channel catfish skin). Tetrahymena induced a similar upregulation, except that HLP-1 was not significantly induced and the response appeared to be more transient than FCA immunostimulation. AMPP levels were increased up to five-fold higher than resting levels and levels expressed were well within concentrations known to be inhibitory to many important pathogens in vitro. These results provide encouragement that AMPP upregulation may be a promising tool in aquaculture for enhancing the resistance of fish to disease.

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream.

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.

Isolation and characterization of a novel myoactive tetradecapeptide-related peptide isolated from the brain of the squid, Todarodes pacificus.

A new bioactive tetradecapeptide, GFKDNVSNRIAHGFamide, was isolated from the brain of the squid, Todarodes pacificus. Using isolated T. pacificus esophagus as a bioassay, the peptide was shown to induce potent contraction of smooth muscle. The threshold concentration for contraction was 5×10(-10) M to 1×10(-9) M. The peptide was homologous to other molluskan (class Gastropoda) and annelid myoactive tetradecapeptides and to some extent, to arthropodan tridecapeptides. A full-length cDNA encoding the biosynthetic precursor of the active peptide was cloned, revealing that the peptide is probably secreted following processing of a prepropeptide containing a signal peptide and prosequences. This is the first myoactive tetradecapeptide (MATP) to be isolated from any mollusk of the class Cephalopoda and we have named it Todarodes tetradecapeptide (TTP).

Primary structure and cellular localization of callinectin, an antimicrobial peptide from the blue crab.

We report the complete amino acid sequence of callinectin, a 32 amino acid, proline-, arginine-rich antimicrobial peptide (AMP) with four cysteines and having the sequence WNSNRRFRVGRPPVVGRPGCVCFRAPCPCSNY-amide. The primary structure of callinectin is highly similar to arasins, AMPs recently identified in the small spider crab (Hyas araneus). Callinectin exists in three isomers that vary in the functional group on the tryptophan (W) residue. The most prevalent isomer had a hydroxy-N-formylkynurenine group, while the other two isomers had either N-formylkynurenine or hydroxy-tryptophan. Using a sequence highly similar to native callinectin, we chemically synthesized a peptide which we called callinectin-like peptide (CLP). Via immuno-electron microscopy, affinity-purified rabbit antibodies raised to CLP successfully localized the site of callinectin in blue crab hemocytes to the large electron-dense granules that are found primarily in large granule hemocytes.

Multiple antibacterial histone H2B proteins are expressed in tissues of American oyster.

We have previously identified a histone H2B isomer (cvH2B-1) from tissue extracts of the bivalve mollusk, the American oyster (Crassostrea virginica). In this paper, we isolate an additional three antibacterial proteins from acidified gill extract by preparative acid-urea-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Extraction of these proteins from tissue was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Addition of protease inhibitors while boiling resulted in somewhat lower yields, with one protein being totally absent with this method. Via mass spectrometry, the masses of one of these purified proteins was 13607.0Da (peak 2), which is consistent with the molecular weight of histone H2B. In addition, via western-blotting using anti-calf histone H2B antibody, all three proteins were positive and were thus named cvH2B-2, cvH2B-3 and cvH2B-4. The antibacterial activity of cvH2B-2 was similar to that of cvH2B-1, with activity against a Gram-positive bacterium (Lactococcus lactis subsp. lactis; minimum effective concentration [MEC] 52-57µg/mL) but inactive against Staphylococcus aureus (MEC>250µg/mL). However, both proteins had relatively potent activity against the Gram-negative oyster pathogen Vibrio parahemolyticus (MEC 11.5-14µg/mL) as well as the human pathogen Vibrio vulnificus (MEC 21.3-25.3µg/mL). cvH2B-3 and cvH2B-4 also had similarly strong activity against Vibrio vulnificus. These data provide further evidence for the antimicrobial function of histone H2B isomers in modulating bacterial populations in oyster tissues. The combined estimated concentrations of these histone H2B isomers were far above the inhibitory concentrations for the tested vibrios, including human pathogens. Our results indicate that the highly conserved histone proteins might be important components not only of immune defenses in oysters but have the potential to influence the abundance of a ubiquitous microbial resident of oyster tissues that is the major source of seafood-borne illness in humans.

Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (Oncorhynchus mykiss) skin and gill.

Antimicrobial polypeptides (AMPPs) are increasingly recognized as a critical component of innate host defense. Among the AMPPs, polypeptides related to histones have been identified from many animals. Using peptide mapping, we further confirm the identity of two histone-like proteins from fish as members of the H2B (sunshine bass) and H1 (rainbow trout) histone groups. We optimized the conditions for measuring rainbow trout HLP-1/H2B via sandwich ELISA. We used two antibodies, one to the amino terminus and one to the carboxyl terminus, of trout histone H2B, as the capture antibodies, and we used peroxidase-labeled antibody raised to calf histone H2B as the secondary antibody. Specificity of the detecting antibody was confirmed by specific reactivity with histone H2B in tissue extracts via western blotting. The test was reproducible and capable of detecting as little as 5 ng of histone H2B (0.05 µg/ml). Histone H2B levels expressed in gill tissue of juvenile, healthy rainbow trout were well within concentrations that are lethal to important fish pathogens. However, there was a significant, age (size)-dependent decline in histone H2B concentrations as fish matured, until levels became virtually undetectable in market-size fish. In contrast, levels in skin appeared to remain high and unchanged in small versus large fish. Antibacterial activity in skin and gill tissues was closely correlated with histone H2B concentration measured via ELISA, which supports our previous finding that histones are the major AMPPs in rainbow trout skin and gill.

Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses.

Antimicrobial polypeptides (AMPPs), consisting of peptides and small proteins with antimicrobial activity, are an integral component of innate immunity. Their often potent properties and widespread prevalence in fish suggests that designing means of manipulating their levels has considerable potential for maintaining or improving fish health. There is evidence that a number of chronic stresses lead to significant downregulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak. Conversely, upregulation of AMPP expression could be used to enhance disease resistance in stressful environments, as well as improve the efficacy of traditional antimicrobial drugs. However, further work is required in linking levels of a number of AMPPs to physiological function since, while a number of studies have documented the down- or upregulation of AMPPs via gene expression, relatively few studies have quantitatively examined changes in protein expression. In addition, not all AMPPs appear to be expressed at microbicidal levels in vivo, suggesting that at least some may have functions other than being directly protective. Nonetheless, in fish, there is evidence that some constitutively expressed AMPPs, such as piscidins and histone-like proteins, are expressed at microbicidal levels and that they decline with stress. Furthermore, certain AMPPs derived from hemoglobin-ß are upregulated to microbicidal levels after experimental challenge. The likely widespread distribution of these three AMPP groups in fish provides the opportunity to design strategies to greatly improve the health of cultured fish populations.

American oyster, Crassostrea virginica, expresses a potent antibacterial histone H2B protein.

An antibacterial protein was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica). Protein isolation was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Adding protease inhibitors while boiling did not have a major effect on activity recovery. In contrast, use of only protease inhibitors (without boiling) resulted in virtually no recovery of this activity. The amino acid sequence of this antibacterial protein was identified as a histone H2B and was designated cvH2B. cvH2B had potent activity against gram-negative bacteria, including the human pathogens Vibrio parahaemolyticus and Vibrio vulnificus, which commonly reside in oyster tissues. We estimated that the concentration of this protein was well within the concentration that was inhibitory to these bacterial pathogens in vitro. This is the first report of the antimicrobial function of histone H2B from any mollusk.

Detection of antimicrobial peptides related to piscidin 4 in important aquacultured fish.

Epithelial surfaces of fish, such as the gut, skin and gills, comprise a large surface area for possible pathogen invasion. Antimicrobial peptides (AMPs), innate immunity components, play a significant role in protecting fish. Piscidins are a family of AMPs. In this study, we detected the presence of the recently discovered piscidin 4 via bug blot, Western blot, ELISA and/or immunohistochemistry in striped bass (Morone saxatilis), white bass (M. chrysops), European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), red drum (Sciaenops ocellatus), and barramundi (Lates calcarifer). Via bug blot, gill extracts from all species had antibacterial activity corresponding to the migration rate of piscidin 4. Western blotting showed that piscidin 4 immunoreactivity was greatest in striped bass gill extract. The concentrations of piscidin 4 detected by the ELISA in striped bass gill (approximately 20 microg/ml) were well within the levels that are inhibitory to important fish bacterial pathogens. Piscidin 4 was also detected via immunohistochemistry in all fish except barramundi. Piscidin 4-positive cells were identified as mast cells (MC), but not all MC were piscidin 4-positive. Species, age, size and physiological condition at sampling were some factors that might affect piscidin expression in different species. Our data provide strong evidence that piscidin 4 isoforms are present in all these commercially important species.

Molecular keys unlock the mysteries of variable survival responses of blue crabs to hypoxia.

Hypoxia is a major stressor in coastal ecosystems, yet generalizing its impacts on fish and shellfish populations across hypoxic events is difficult due to variability among individuals in their history of exposure to hypoxia and related abiotic variables, and subsequent behavioral and survival responses. Although aquatic animals have diverse physiological responses to cope with hypoxia, we know little about how inter-individual variation in physiological state affects survival and behavioral decisions under hypoxic conditions. Laboratory experiments coupled with molecular techniques determined how extrinsic factors (e.g., water body and temperature) and respiratory physiology (hemocyanin concentration and structure) affected survival and behavior of adult blue crabs (Callinectes sapidus) exposed to different levels of hypoxia over a 30-h time period. Nearly 100% of crabs survived the 1.3 mg dissolved oxygen (DO) l(-1) treatment (18.4% air saturation), suggesting that adult blue crabs are tolerant of severe hypoxia. Probability of survival decreased with increasing hypoxic exposure time, lower DO, and increasing temperature. Individual-level differences in survival correlated with water body and crab size. Crabs collected from the oligo/mesohaline and hypoxic Neuse River Estuary (NRE), North Carolina, USA survived hypoxic exposures longer than crabs from the euhaline and normoxic Bogue and Back Sounds, North Carolina. Furthermore, small NRE crabs survived longer than large NRE crabs. Hemocyanin (Hcy) concentration did not explain these individual-level differences, however, hypoxia-tolerant crabs had Hcy structures indicative of a high-O(2)-affinity form of Hcy, suggesting Hcy "quality" (i.e., structure) may be more important for hypoxia survival than Hcy "quantity" (i.e., concentration). The geographic differences in survival we observed also highlight the importance of carefully selecting experimental animals when planning to extrapolate results to the population level.

Immunocytochemical localization of piscidin in mast cells of infected seabass gill.

Annual losses of approximately 5-10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.

Environmental and physiological controls of blue crab avoidance behavior during exposure to hypoxia.

Generalizing the impacts of hypoxia on aquatic animal populations is difficult due to differences in behavioral and physiological responses among individuals as well as varying hydrodynamics of hypoxic events. Information on which environmental cues animals use to avoid hypoxia and how abiotic covariates and physiology influence avoidance behavior is lacking. Our laboratory flume studies quantified the interacting effects of hydrography (dissolved oxygen [DO], temperature, and salinity), hydrodynamics (rate of DO decline and current speed), and physiological state on avoidance behaviors of blue crabs (Callinectes sapidus). Changes in DO stimulated increased rates of movement, regardless of whether the change resulted in hypoxia. Increased rates of DO decline stimulated faster movement rates under hypoxic conditions because crabs spent less time in hypoxia compared to crabs under conditions of slow rate of DO decline. Blue crabs that had hemocyanin structures with a high affinity for O(2) (hypoxia-tolerant) were less active under hypoxic conditions than conspecifics with hemocyanins with a low O(2) affinity, suggesting that physiological state influences behavioral responses to stressors. These results provide a mechanistic understanding of how physiological acclimation and hypoxia hydrodynamics may influence population dynamics.

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides.

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18-26 amino acids long.We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male).We have named this peptide "piscidin 4" since it has considerable (to >65%) N-terminal sequence homology to piscidins 1-3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.