Epidermal Mucus Research Articles

The Toxins of Nemertean Worms.

Most ribbon worms (phylum: Nemertea) are found in marine environments, where they act as predators and scavengers. They are characterized by an eversible proboscis that is used to hunt for prey and thick mucus covering their skin. Both proboscis and epidermal mucus mediate toxicity to predators and preys. Research into the chemical nature of the substances that render toxicity has not been extensive, but it has nevertheless led to the identification of several compounds of potential medicinal use or for application in biotechnology. This review provides a complete account of the current status of research into nemertean toxins.

Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly.

Healthcare-associated infections resulting from bacterial attachment and biofilm formation on medical implants are posing significant challenges in particular with the emergence of bacterial resistance to antibiotics. Here, we report the design, synthesis and characterization of self-assembled nanostructures, which integrate on their surface antibacterial peptides. The antibacterial WMR peptide, which is a modification of the native sequence of the myxinidin, a marine peptide isolated from the epidermal mucus of hagfish, was used considering its enhanced activity against Gram-negative bacteria. WMR was linked to a peptide segment of aliphatic residues (AAAAAAA) containing a lipidic tail (C19H38O2) attached to the ε-amino of a terminal lysine to generate a peptide amphiphile (WMR PA). The self-assembly of the WMR PA alone, or combined with coassembling shorter PAs, was studied using spectroscopy and microscopy techniques. The designed PAs were shown to self-assemble into stable nanofiber structures and these nanoassemblies significantly inhibit biofilm formation and eradicate the already formed biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Candida albicans (pathogenic fungus) when compared to the native WMR peptide. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity, and establish an innovative strategy to develop self-assembled antimicrobial materials for biomedical applications.

Antimicrobial Activity of Epidermal Mucus from Top Aquaculture Fish Species against Medically-Important Pathogens

Zoonotic infections caused by bacterial pathogens are considered as major threat to humans and the aquaculture industry. This problem triggered the search for various natural products from plants, microorganisms, animal tissues, and secretions to determine the presence of metabolites that may be of potential antimicrobial effects against infectious agents. However, limited attempts have been conducted to elucidate the potential use of freshwater fish mucus in against pathogens. Here, the antimicrobial activity of mucus of economically-important freshwater fish species in the Philippines: Oreochromis niloticus (tilapia), Clarias batrachus (catfish), and Channa striata (snakehead fish) was investigated against fish and human pathogens. The pooled fish mucus was extracted with succeeding centrifugation and filtration. The acidic mucus extracts were tested for antimicrobial-inhibitory effects and minimum inhibitory concentration (MIC) by agar-overlay diffusion and microbroth plate dilution method, respectively. The results showed that all fish mucus extracts exhibited antimicrobial effects against test pathogens with catfish exhibiting the highest inhibitory effects against Pseudomonas aeruginosa (p = 0.096), Klebsiella pneumoniae (p = 0.000), Enterococcus faecalis (p = 0.665), Micrococcus luteus (p = 0.000), Aeromonas hydrophila (p = 0.000), Staphylococcus aureus (p = 0.000), Escherichia coli (p = 0.000), and Serratia marcescens (p = 0.000) as compared to the broad-spectrum antibiotic control, Cefoperazone. Interestingly, catfish mucus revealed inhibitory effects against Gram-positive S. aureus and M. luteus at the lowest concentration (1:4 dilution). The present findings revealed the potential antimicrobial use of freshwater fish mucus against medically-important pathogens.

Antibacterial Activity of the Epidermal Mucus of Barbodes everetti

The epidermal mucus of fish contains antimicrobial agents that act as biological defence against disease. This study aims to identify antibacterial activity and protein concentration of epidermal mucus of Barbodes everetti, a Bornean endemic freshwater fish. The epidermal mucus was extracted with 3% acetic acid, 0.85% sodium chloride and crude solvents. The mucus activity against eight strains of human pathogenic bacteria, including Bacillus cereus ATCC 33019, Escherichia coli O157:H7, Listeria monocytogenes ATCC 7644, Pseudomonas aeruginosa ATCC 27853, Salmonella braenderup ATCC BAA 664, Salmonella typhimurium, Staphylococcus aureus ATCC 25933, and Vibrio cholerae, were tested. The acetic acid mucus extract of B. everetti was able to inhibit five strains of bacteria and show no activity toward E. coli O157:H7, B. cereus ATCC 33019 and L. monocytogenes ATCC 7644. Moreover, the highest protein concentration was quantified in crude extract, followed by aqueous and acetic acid extracts. This study provides a preliminary knowledge on the activity of epidermal mucus of B. everetti towards five out of the eight human pathogens tested, therefore it may contain potential sources of novel antibacterial components which could be further extracted for the production of natural antibiotics towards human-related pathogenic bacteria.
 Keywords: Antibacterial, antimicrobial properties, Barbodes everetti, epidermal mucus, pathogenic bacteria

Estimating stable isotope turnover rates of epidermal mucus and dorsal muscle for an omnivorous fish using a diet-switch experiment

Stable isotope (SI) analysis studies rely on knowledge of isotopic turnover rates and trophic-step discrimination factors. Epidermal mucus (‘mucus’) potentially provides an alternative SI ‘tissue’ to dorsal muscle that can be collected non-invasively and non-destructively. Here, a diet-switch experiment using the omnivorous fish Cyprinus carpio and plant- and fish-based formulated feeds compared SI data between mucus and muscle, including their isotopic discrimination factors and turnover rates (as functions of time T and mass G, at isotopic half-life (50) and equilibrium (95)). Mucus isotope data differed significantly and predictively from muscle data. The fastest δ13C turnover rate was for mucus in fish on the plant-based diet (T_{50}: 17 days, T_{95}: 74 days; G_{50}: 1.08(BM), G_{95}: 1.40(BM)). Muscle turnover rates were longer for the same fish (T_{50}: 44 days, T_{95}: 190 days; G_{50}: 1.13(BM), G_{95}: 1.68(BM)). Longer half-lives resulted in both tissues from the fish-based diet. δ13C discrimination factors varied by diet and tissue (plant-based: 3.11–3.28‰; fishmeal: 1.28–2.13‰). Mucus SI data did not differ between live and frozen fish. These results suggest that mucus SI half-lives provide comparable data to muscle, and can be used as a non-destructive alternative tissue in fish-based SI studies.

Skin mucus metabolites in response to physiological challenges: A valuable non-invasive method to study teleost marine species

Knowledge concerning the health and welfare of fish is important to conserve species diversity. Fish mucosal surfaces, and particularly the skin, are of utmost importance to protect the integrity and homeostasis of the body and to prevent skin infections by pathogens. We performed three trials simulating different environmental and anthropogenic challenges: fish capture (air exposure), bacterial infection and fasting, with the aim of evaluating epidermal mucus as a non-invasive target of studies in fish. In this initial approach, we selected three well-known marine species: meagre (Argyrosomus regius), European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) for our study. Mucus viscosity was measured in order to determine its rheological properties, and mucus metabolite (glucose, lactate, protein and cortisol) levels were analysed to establish their suitability as potential biomarkers. Skin mucus appeared as a viscous fluid exhibiting clearly non-Newtonian behaviour, with its viscosity being dependent on shear rate. The highest viscosity (p < 0.05) was observed in sea bream. Mucus metabolites composition responded to the different challenges. In particular, glucose increased significantly due to the air exposure challenge in meagre; and it decreased during food deprivation in sea bream by a half (p < 0.05). In contrast, mucus protein only decreased significantly after pathogenic bacterial infection in sea bass. In addition, mucus lactate immediately reflected changes closely related to an anaerobic condition; whereas cortisol was only modified by air exposure, doubling its mucus concentration (p < 0.05). The data provided herein demonstrate that mucus metabolites can be considered as good non-invasive biomarkers for evaluating fish physiological responses; with the glucose/protein ratio being the most valuable and reliable parameter. Determining these skin mucus metabolites and ratios will be very useful when studying the condition of critically threatened species whose conservation status prohibits the killing of specimens.

Crude extracts of epidermal mucus and epidermis of climbing perch Anabas testudineus and its antibacterial and hemolytic activities

Epidermal mucus of fish contains a number of biologically active constituents including antimicrobial peptides that are continuously expressed and also engaged in provision of protection to the fish against injurious substances as well as potential pathogenic microbes. This study seeks to determine the hemolytic and antibacterial properties of crude extract from epidermal mucus of climbing perch. Epidermal mucus and epidermis were obtained from 15 climbing perches after hypothermic stress stimulation for extraction of crude by aqueous and acidic methods. Antibacterial activity by well diffusion agar was applied for both extracts against 11 strains belonging to 7 different bacterial species using well agar diffusion test assay. In addition, haemolytic activity was investigated using horse RBCs. Antibacterial activity was performed for acidified crude extract only and it was found to be significant (P < 0.05) against P. aeruginosa while the least reactivity was observed against Methicillin Resistant Staphylococcus aureus. Total soluble protein concentration was 1.2 mg/ml with 13 bands of proteins (245–11) kDa distinguished. There was absence of haemolytic activity of acidified crude extract using horse RBCs. The finding of this study indicates that proteins in bioactive crude extracted from climbing perch have potential therapeutic application.

Peptide ion channel toxins from the bootlace worm, the longest animal on Earth

Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the α-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the ‘bootlace worm’ known as the longest animal on earth. The most abundant peptide, the 31-residue long α-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight α-nemertides, mainly distributed in the genus Lineus. α-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 µg/kg (~300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Nav1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that α-nemertides can be promising candidates for development of bioinsecticidal agents.

Selective ingestion contributes to the stoichiometric homeostasis in tissues of the endogeic earthworm Aporrectodea turgida

Most detritivores maintain a stoichiometric homeostasis in their body tissue regardless of the chemical composition of the substrates they ingest, but the mechanisms for stoichiometric regulation in soil-inhabiting detritivores like earthworms are poorly understood. The objectives of this study were (1) to examine whether the endogeic earthworm Aporrectodea turgida (Eisen) exhibits a strict homeostasis in its tissue C:N ratio, (2) to determine if A. turgida controlled its tissue N concentration by changing the quantity of N excreted in epidermal mucus and (3) to consider how the gut transit time, gut load and cast production were related to selective ingestion, which is hypothesized to control the N stoichiometry in A. turgida tissues. Two laboratory experiments were designed to address these objectives. In the first experiment, we evaluated the C and N concentrations, and C:N ratio of A. turgida body tissue and epidermal mucus after the earthworm fed on soil mixed with 15N-labeled plant litter (red clover leaves, wheat leaves, wheat stem) having variable N content and C:N ratios, as well as no litter, for 7 days. The second experiment measured the gut transit time, gut load and cast production of A. turgida fed soil marked with glass beads, either without litter or mixed with plant litter (soybean leaves with high N, wheat stems with low N). The endogeic earthworm A. turgida maintained strict homeostasis in their body tissue, with a C:N ratio of 3.9. The epidermal mucus of A. turgida also showed a strict homeostasis (C:N ratio = 4.6) and constant 15N enrichment, regardless of the N content in plant litter. Therefore, N secretion through epidermal mucus cannot be a mechanism that regulates the N stoichiometry in the body tissue of A. turgida. The gut transit time of ingested substrates was the same, as both N-rich (i.e., soil-soybean mixture) and N-poor (i.e., soil-wheat mixture) substrates took 21 ± 1 h to pass from the mouth to the anus of A. turgida, however, there was significantly (P < 0.05) less material in the gut and less cast production from the N-rich than the N-poor substrate. We conclude that a selective ingestion process controls the intake of organic substrates and likely contributes to the conservation of N stoichiometry in A. turgida body tissues.

Mukus Epidermis Clarias Batrachus Merencat Pertumbuhan Bakteria Gram Negatif

Recently, epidermal mucus of fish has been identified as a rich biological source of various bioactive substances, particularly antibacterial peptides. This study was conducted to identify the potential of epidermal mucosa of Clarias batrachus as a source of antibacterial material. Mucosal samples were collected from C. batrachus epidermis and extracted using an aqueous extraction method. Antibacterial activity of the crude epidermal mucus extract was determined by using spectrophotometric method. As a result there was antibacterial activity by the mucus extract against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The results of this study supports the role of mucus as a part of body immune system with antibacterial peptide as a component of innate body immune system. Hence, these findings can be used as one of the first steps towards the development of new class antibiotics. DOI : http://dx.doi.org./10.17576/JSKM-2018-12

Mukus Epidermis Clarias Batrachus Merencat Pertumbuhan Bakteria Gram Negatif

Recently, epidermal mucus of fish has been identified as a rich biological source of various bioactive substances, particularly antibacterial peptides. This study was conducted to identify the potential of epidermal mucosa of Clarias batrachus as a source of antibacterial material. Mucosal samples were collected from C. batrachus epidermis and extracted using an aqueous extraction method. Antibacterial activity of the crude epidermal mucus extract was determined by using spectrophotometric method. As a result there was antibacterial activity by the mucus extract against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The results of this study supports the role of mucus as a part of body immune system with antibacterial peptide as a component of innate body immune system. Hence, these findings can be used as one of the first steps towards the development of new class antibiotics. DOI : http://dx.doi.org./10.17576/JSKM-2018-12

Epidermal mucus, a major determinant in fish health: a review.

Fish epidermal mucus contains innate immune components, secreted by globlet cells that provide the primary defence against different pathogenic microbes and act as a barrier between fish and its immediate niche. The major function of mucus includes entrapment and sloughing of microbes. The mucus also contains many factors such as antimicrobial peptides (AMPs), lysozymes, lectins, proteases, etc that provide innate immunity. The AMPs secreted by epidermal mucus cells displayed antimicrobial activity against a variety of pathogens. Besides, mucosal lysozyme was found to produce significant bacteriolytic action whereas different proteases found in skin mucus of fish can kill the pathogens by cleaving its protein or by activating immunological mechanisms. Lectins are also mucosal agglutinins that play a diverse role in innate immunity like opsonization, activation of complement, etc. Epidermal mucus in fish thus provides an innate and fast acting protection which is non-specific and is found to be relatively temperature independent. The aim of the present review is to provide a broad overview of the different components of epidermal mucus including AMPs, proteases, lysozymes as well as their mode of action on pathogens.

Exposure to synthetic hydraulic fracturing waste influences the mucosal bacterial community structure of the brook trout (&lt;em&gt;Salvelinus fontinalis&lt;/em&gt;) epidermis

Production of natural gas using unconventional technologies has risen as demand for alternative fuels has increased. Impacts on the environment from waste generated from these processes are largely unexplored. In particular, the outcomes of organismal exposure to hydraulic fracturing waste have not been rigorously evaluated. We evaluated the effects of exposure to surrogate hydraulic fracturing waste (HF waste) on mucosal bacterial community structure of the brook trout (Salvelinus fontinalis) epidermis. Brook trout are fish native to streams at risk to HF waste exposure. Here, fish were exposed to four treatments (control, 0.00%; low, 0.01%; medium, 0.10%; and high, 1.0% concentrations) of surrogate HF waste synthesized to mimic concentrations documented in the field. Epidermal mucus samples were collected and assessed 15 days post-exposure to determine if the associated bacterial community varied among treatments. We observed differences in epidermal mucosal bacterial community composition at multiple taxonomic scales among treatments. These community changes reflected compositional differences in taxa dominance and community similarity rather than losses or gains in taxonomic richness. The dominant bacterial genus that explained the greatest variation in community structure between exposed and unexposed fish was Flavobacterium. Two genera associated with salmonid diseases, Flavobacterium and Pseudomonas, were statistically more abundant in high treatments than controls. These results suggest that exposure to low levels of HF waste influences bacterial colonization and may lead to a disruption that favors bacterial populations associated with fish disease.

Diet of invasive pikeperchSander lucioperca: developing non-destructive tissue sampling for stable isotope analysis with comparisons to stomach contents analysis

Impact assessments of invasive piscivorous fishes usually rely on dietary analyses to quantify their predation pressure on prey communities. Stomach contents analysis (SCA), typically a destructive sampling method, is frequently used for this. However, many invasive piscivores are exploited by catch-and-release sport angling, with destructive sampling often not feasible. Stable isotope analysis (SIA) provides an alternative dietary analysis tool to SCA, with use of fin tissue, scales and/or epidermal mucus potentially enabling its non-destructive application. Here, the diet of a population of pikeperchSander lucioperca, an invasive sport fish to Great Britain, was investigated by applying SIA to a range of tissues. Testing SI data of dorsal muscle (destructive sampling) versus fin, scale and mucus (non-destructive sampling) revealed highly significant relationships, indicating that the tissues collected non-destructively can be reliably applied to pikeperch diet assessments. Application of these SI data to Bayesian mixing models predicted that asS. luciopercalength increased, their diet shifted from macro-invertebrates to fish. Although similar ontogenetic patterns were evident in SCA, this was inhibited by 54% of fish having empty stomachs. Nevertheless, SCA revealed that asS. luciopercalength increased, their prey size significantly increased. However, the prey:predator length ratios ranged between 0.08 and 0.38, indicating most prey were relatively small. These results suggest that when non-destructive sampling is required for dietary analyses of sport fishes, SIA can be applied using fin, scales and/ or mucus. However, where destructive sampling has been completed, SCA provides complementary dietary insights, especially in relation to prey size.

Comparative Immunological and Biochemical Properties of the Epidermal Mucus from Three Brackishwater Fishes

Immunological and biochemical properties of skin mucus of different fish species may have mutual beneficial effects on health management when the fishes are farmed together in same system. Present experiment was conducted to investigate and compare the immunological and biochemical properties of epidermal mucus of three brackishwater fishes, namely Lates calcarifer, Chanos chanos and Mugil cephalus. Mucus was collected from the dorso-lateral surface of six individual fish of each species and used for analysis of immunological properties and biochemical composition. Innate immune parameters such as lysozyme, haemolytic, phagocytic activities and lectin were significantly (p < 0.05) more prominent in the mucus of C. chanos followed by that of L. calcarifer and M. cephalus. Similarly, mucus of C. chanos exhibited maximum protease, alkaline phosphatase, and antibacterial activities. UV spectral analysis showed the presence of toluene, isoquinoline, 2-furaldehyde, octadecenoic acid, biphenyl, thymidine, and cinnamic acid in the mucus of L. calcarifer, whereas these were absent in other two species. Fourier transform-infrared spectrum analysis revealed that isothiocyanate, aldehyde and alkene were common functional groups in the mucus of all three fishes. The results indicated that mucus of C. chanos has stronger innate immune properties as compared to that of other two fishes and therefore, polyculture of this fish with other fish or shrimp species may have beneficial effects for disease prevention.

Use of epidermal mucus in elasmobranch stable isotope studies: a pilot study using the giant manta ray (Manta birostris)

Stable isotope analysis of tissues with different turnover rates allows for a broader temporal view of a species’ feeding habits. Epidermal mucus is a rapid to medium turnover ‘tissue’ in teleost fish, but its use in elasmobranch dietary studies is unknown. In the present study, we conducted stable isotope analysis on mucus and muscle from the giant manta ray Manta birostris. Mucus δ13C values were depleted and closer to surface zooplankton δ13C values compared with muscle, whereas there was no significant difference in δ15N values between these two tissue types. Using diet tissue discrimination factors from the literature, there was no significant difference in the expected prey profile between muscle and epidermal mucus. However, a broader range in δ13C values of expected prey was shown for mucus compared with muscle. The results suggest that if M. birostris mucus is indicative of recent dietary intake, resource use during aggregative behaviour off Ecuador is broader, but with no obvious resource switching. The present study is the first example of using bulk stable isotope analysis to evaluate mucus to investigate feeding ecology in elasmobranchs. However, the time course for the change in mucus isotope signature still needs to be determined through controlled feeding studies in an aquarium setting.

Comparison of biochemical and cytotoxic activities of extracts obtained from dorsal spines and caudal fin of adult and juvenile non-native Caribbean lionfish (Pterois volitans/miles)

Pterois volitans/miles lionfish (adult and juvenile) dorsal spines and caudal fin extracts were compared in their general composition, enzymatic activities and hemolytic and cytotoxic effects on bovine aortic endothelial cells and murine myoblasts, to distinguish between the activities present in the venom and epidermal mucus. Intradermal and intramuscular injections were also administered in mice to determine in vivo effects.This work shows that crude venom of Caribbean species of lionfish, present in dorsal spines, induces several in vitro effects including hemolysis, weak cytotoxicity, proteolytic and hyaluronidase activities, whereas in vivo, it is not hemorrhagic nor myotoxic, but causes edema, plasma extravasation and a thrombotic-associated lesion on the skin. Some small differences were observed between adult and juvenile venomous secretions. Gelatinolytic activity of the epidermal mucus, the only activity found in caudal fin extracts, could contribute to the in vivo toxicity of the venom.

Survey of Antibiotic-producing Bacteria Associated with the Epidermal Mucus Layers of Rays and Skates.

Elasmobranchs represent a distinct group of cartilaginous fishes that harbor a remarkable ability to heal wounds rapidly and without infection. To date very little work has addressed this phenomenon although it is suggested that antibiotic capabilities associated with epidermal surfaces may be a factor. The study of benefits derived from mutualistic interactions between unicellular and multicellular organisms is a rapidly growing area of research. Here we survey and identify bacterial associates of three ray and one skate species in order to assess the potential for antibiotic production from elasmobranch associated bacteria as a novel source for new antibiotics.

Isotopic analysis of epidermal mucus in freshwater fishes can reveal short‐time diet variations

AbstractAlthough stable isotope analysis is a powerful tool for determining animal diets, migration patterns, and the structure of food webs in aquatic systems, the slow response of isotopic ratios in the traditionally used muscle tissue of fishes often hampers this approach. By analyzing tissues with more rapid isotopic change, this technique can be used over a finer time scale than traditional isotopic analyses. We conducted a diet‐switch experiment to compare changes in the stable carbon and nitrogen isotope ratio values (δ13C and δ15N, respectively) in epidermal mucus, fin and muscle tissues of three species of freshwater cyprinid fishes. The half‐life of the isotopic change ranged from 62 to 144 days in mucus, 57 to 202 days in fin, and 196 to 680 days in the muscle tissue. The isotope ratios in mucus changed more rapidly than those in muscle tissues in the three species examined, similarly to the previously studied three species (catfish, steelhead, freshwater goby), whereas fin showed relatively variable trends in isotopic change. Interspecific variation in the trophic discrimination factor (TDF) was particularly large in the δ13C values of fin tissue compared with those in the other two tissues. With generally rapid change and relatively invariable TDF, the use of epidermal mucus in isotope analysis instead of the widely used fin and muscle tissues is advantageous for studying short‐term changes in food habits of fishes and food web structures.

Intensive epidermal adsorption and specific venous deposition of carboxyl quantum dots in zebrafish early-life stages

To properly assess the environmental risk of quantum dots (QDs), it is necessary to determine their fate in living organisms, including adsorption, distribution and bioaccumulation under representative environmental or physiological conditions. We comprehensively investigated the fate of QDs with carboxyl terminal functional groups (carboxyl-QDs) in zebrafish (Danio rerio) embryo and larvae subjected to either waterborne exposure or cardiovascular system microinjection. On waterborne exposure, carboxyl-QDs exhibited an intensive adsorption and accumulation in the chorion of embryos, and their predominate target organs were the gill and intestinal tract in larvae. On microinjection, carboxyl-QDs were rapidly delivered into the cardiovascular system and specifically deposited in veins and the capillary network system of zebrafish larvae, but not in the arterial system. Taken together, we found that the exact tissue condition including epidermal structures, mucus secretion and vascular microstructures strongly affected the adsorption, uptake and distribution of carboxyl-QDs in zebrafish. This work highlights the intensive tissue epidermal adsorption and accumulation of carboxyl-QDs and their specific vein and capillary deposition in the cardiovascular system in zebrafish early-life stages.