Description and comparative study of physico-chemical parameters of the teleost fish skin mucus

Abstract

The study of mucosal surfaces, and in particular the fish skin and its secreted mucus, has been of great interest recently among immunologists. Measurement of the viscosity and other physico-chemical parameters (protein concentration, pH, conductivity, redox potential, osmolality and density) of the skin mucus can help to understand its biological functions. We have used five marine species of teleost: gilthead seabream (Sparus aurata L.), European sea bass (Dicentrarchus labrax L.), shi drum (Umbrina cirrosa L.), common dentex (Dentex dentex L.) and dusky grouper (Epinephelus marginatus L.), all of them with commercial interest in the aquaculture of the Mediterranean area. Mucus showed a direct shear- and temperature-dependent viscosity, with a non-Newtonian behavior, which differed however between two groups: one with higher viscosity (D. labrax, U. cirrosa, D. dentex) and the other with lower viscosity (S. aurata, E. marginatus). In addition, there was a clear interrelation between density and osmolality, as well as between density and temperature. Taking into account that high values of viscosity should improve the barrier effect against pathogens but low values of viscosity are needed for good locomotion characteristics, our results may help elucidate the relationship between physico-chemical and biological parameters of skin mucus, and disease susceptibility.

Keywords

Fish viscosity protein concentration density osmolality teleosts skin mucus

1. Introduction

Vertebrate mucus is a viscous colloid gel that forms an adherent layer that covers the epithelium [1], such as skin and gut, and is secreted by various epithelial cells such as goblet cells found in mucous glands [2,3]. In the fish skin, mucus plays a critical role as a natural, semipermeable, dynamic, physical, chemical and biological barrier [4,5] that allows exchange of nutrients, water, gases, odorants, hormones and gametes [6]. Skin mucus is the part of the fish which is contact with surrounding water and, as a consequence the resistance to locomotion through the water could be influenced by the variation in its rheology. The range of roles for fish mucus is very large and includes respiration, ionic and osmotic regulation, reproduction, locomotion, defence against microbial infections, disease resistance and protection, excretion and communication [2,7]. Apart from acting as a physical barrier, the abundance of substances with immune functions has highlighted important immunological properties of fish skin mucus, including cytokines [8], antimicrobial peptides [9,10], lysozyme [10,11], lipoprotein [12], complement factors [13], lectins [14–16], proteases [11,17] and antibodies [11,18–20]. Some components only have a defensive purpose [21], whereas others may also act by modifying the organization and properties of the gel [22].

Mucus is primarily composed of water (approx. 95%) but also contains salts, lipids (e.g. fatty acids, phospholipids, cholesterol) and glycoproteins (mucins) with molecular weights ranging from 0.5 to 20 MDa [23,24]. Mucins are the main components of fish skin mucus, responsible for its viscous and elastic gel-like properties, and exert a mechanical barrier by serving as filters for pathogens and preventing pathogen adherence to the underlying tissues [25], also forming a matrix in which a diverse range of antimicrobial molecules can be found [26]. These macromolecules are heavily glycosylated filamentous proteins that can form gel or non-gel structures. Moreover, they are strongly adhesive, and play a major role in the defence of the mucosae [21,27] by providing appropriate viscoelastic and rheological properties to mucosal layers [22]. The carbohydrate side chains constitute up to 80% of the total mucin mass [21,22] and give an elongated and rigid structure to the molecules, which contributes to these properties [28]. The proteinaceous part of the mucin typically possess repetitive regions rich in threonine, serine and, to a lesser extent, proline; these are the sites where glycosylation takes place [29]. Due to high levels of glycosylation, many functions of the mucins depend on their carbohydrate chains, which offer wide possibilities of interactions with the environment in addition to participating in the mechanical properties of the mucus [21]. Given that mucus is supposed to play so many roles on the fish surface, it is surprising that the scientific literature includes few measurements of the physical and chemical properties, which are essential for proper biological functions. Advances have been made in identifying and characterizing the major oligomeric fish mucins [30,31], but, no studies related their physical or chemical properties to a specific set of rheological parameters for a mucus gel. Thus, the aim of the present study was to investigate the physico-chemical parameters of the skin mucus of different species of marine teleosts, to gain a better understanding of the biology and function of this essential barrier.

2. Materials and methods

2.1. Fish care and maintenance

Thirty adult specimens of each of the following species were sampled in June 2013 at the Instituto Español de Oceanografía (IEO, Mazarrón, Spain) installations: gilthead seabream (Sparus aurata L.) (125 ± 25 g body weight), European sea bass (Dicentrarchus labrax L.) (100 ± 18 g body weight), shi drum (Umbrina cirrosa L.) (565.5 ± 51 g body weight), common dentex (Dentex dentex L.) (1,600 ± 210 g body weight) and dusky grouper (Epinephelus marginatus L.) (803 ± 106 g body weight). All fish species were bred and kept at the IEO facilities except the groupers that were caught at the juvenile stage from the wild and reared in the same facilities for more than 3 years. The fish were kept in 2 m³ tanks with a flow-through circuit (density 5–10 kg biomass m⁻³), suitable aeration, filtration system, natural photoperiod and water temperature (14.6–17.8°C). All the rearing conditions were the same for all species and fish were sampled at the same time to avoid changes due to different salinity, temperature, handling, feeding, photoperiod, etc. The environmental parameters, mortality and food intake were recorded daily.

2.2. Skin mucus collection

Fish were anesthetized prior to sampling with 100 mg l⁻¹ MS222 (Sandoz). Skin mucus samples were collected according to the method of Guardiola et al. [11]. Briefly, the dorso-lateral surface of naïve specimens was gentle scraping by using a cell scraper, with enough care to avoid contamination with blood and/or urino-genital and intestinal excretions. In order to get enough mucus for all the assays, equal samples of mucus were pooled (1 pool of 30 fish) and stored at −80°C.

2.3. Physico-chemical parameters

Protein concentration in skin mucus samples was determined by the Bradford method [32]. The pH measurements were done by a pH & ION-Meter GLP 22+ (Crison). Conductivity measurements were carried out at 25°C using a Crison Conductimeter microCM 2200 and compared with the reference solution (ÉTALON CONDUCTIVITÉ 97 10 of 1,413 µS cm⁻¹; Crison). Redox potential values were obtained from potentiometric measurements carried out with an electrode system consisted of a Crison platinum electrode 52-67 and an Orion Ag/AgCl double-junction reference electrode (Orion 90-02) connected to a homemade high-impedance data acquisition device connected to a personal computer. Redox standard solutions of 124 and 250 mV (Fluka) were used as reference.

Osmolality was measured using a VAPRO vapor pressure osmometer (model 5520). Density measurements were made in a densitometer (MDA5000M Anton Paar) at 12, 17 and 22 ± 0.1°C (the typical range of temperature variation between winter and summer in the Mediterranean Sea). In this instrument the sample is introduced into a U-shaped borosilicate glass tube that is excited to vibrate at a characteristic frequency. Density is determined by the frequency changes due to the presence of the sample. In order to estimate the reproducibility of measurements, six different repetitions were performed for samples, giving a standard deviation of 0.002 g l⁻¹.

2.4. Mucus viscosity

For viscosity analysis, samples were thawed on ice, briefly vortexed and centrifuged at 7,000 g for 5 min (MiniSpin, Eppendorf). The supernatants were collected, measured for their viscosity and the remaining mucus was stored at −20°C for later analysis. Viscosity measurements were made in two different devices. For lower shear rates we used a plate–plate rheometer (Anton Paar MCR 102; plate model PP50, plate–plate distance 0.8 mm) using approximately 1.6 ml of sample aliquots at 12, 17 and 22 ± 0.1°C. To obtain a characteristic profile of viscosity, it was measured over a range of shear rates (11.5, 23, 46 and 115 s⁻¹) at a constant shear rate for 200 s, recording values every 1 second. During analysis, the first 20 points were removed in order to eliminate artefacts at the beginning of the measurement. To obtain stable recording, mucus samples were allowed to equilibrate for 5 min after each measurement and before a new measurement was made.

We also used a Rolling Ball microviscometer (Lovis 2000ME, Anton Paar) to obtain shear rates of >400 s⁻¹. Samples were treated as above and aliquots of 1 ml were used for measurement at 12, 17 and 22 ± 0.1°C. In this instrument a ball rolls through a closed capillary tube, which is filled with the sample and is inclined at a defined angle. Changing the angle, one can submit the sample to different shear rates. Three inductive sensors in the tube determine the rolling time between defined marks, and sample viscosity is directly proportional to the rolling time. All shear rates were slightly above 400 s⁻¹.

3. Results

Our main objective was to characterize the viscosity of mucus for all species studied. We have added the measurement of some other properties, first, to find differences between different mucus and second, and more important in the context of this work, to obtain some other physico-chemical parameters that could support and extend the interpretation of the viscosity measurements. Firstly, viscosity values (Fig. 1) broadly fell into two groups, one with higher viscosity (D. labrax, U. cirrosa, D. dentex) and the other with lower viscosity (S. aurata, E. marginatus). In addition, mucus viscosity for all fish species showed a non-Newtonian behaviour, i.e. a shear-thinning effect where mucus exhibited a greater viscosity at low shear rates than at high ones (Fig. 1). The greatest differences were found from the lowest shear rate (11.5 s⁻¹) and temperature (12°C) (Fig. 1(a)), whilst slight differences were found in the higher shear rates and temperature tested (22°C) (Fig. 1(c)). Water shows a Newtonian behaviour and the viscosity values were 1.234, 1.0798 and 0.9544 mPa · s at 12, 17 and 22°C, respectively. The viscosity variation at high shear due to temperature is quantitatively very similar for skin mucus and water. If we take into account all samples studied, at high shear rate we find values for viscosity of 1.8 ± 0.2 vs 1.5 from 12 to 17°C and 1.5 ± 0.3 vs 0.13 from 17 to 22°C.

Fig. 1.

Viscosity (mPa · s) in relation to shear rate (s⁻¹) in skin mucus of S. aurata, D. labrax, U. cirrosa, D. dentex and E. marginatus specimens at 12, 17 and 22 ± 0.1°C. Data represent the value of a pool of 30 fish ± SEM of the technical replicates.

Other physico-chemical parameters such as protein concentration, pH, conductivity, redox potential, osmolality (Table 1) and density (Table 2) were measured in the skin mucus from all marine fish under study. Protein concentration was similar in all fish species, with the highest and the lowest recorded values obtained for mucus of D. labrax and S. aurata, respectively. The pH was also very similar among species, being slightly lower for D. labrax. Regarding conductivity, the highest and the lowest values were obtained for skin mucus of S. aurata and U. cirrosa, respectively. Skin mucus redox potential was similar in S. aurata, U. cirrosa and D. dentex and lowest in D. labrax. The highest osmolality value was obtained in the skin mucus of S. aurata and the lowest value in D. labrax samples, compared to mucus from the other tested fish species (Table 2). Finally, as expected, a decrease in mucus density was observed with increasing temperature in all species, with the greatest reduction in S. aurata and the lowest in D. labrax (Table 2).

4. Discussion

The fish skin mucus forms a physico-chemical barrier that protects epithelial surfaces in many different ways. However, most studies have only focused on its immunological functions since it is the first line of defence against microorganisms and contains many immune factors that protects fish against infections [25,33], and whose secretion is modulated by the microenvironment, neural, endocrine and immune factors [26]. These immune components of fish mucus have been studied in several species including the marine fish species tested in the present study [34]. Nevertheless, information about physico-chemical parameters of fish skin mucus that might be correlated with its biological properties, is not available. Strikingly, to our knowledge, there are only two studies evaluating these parameters in the skin mucus of teleosts [35,36].

Table 1

Physico-chemical parameters of skin mucus from S. aurata, D. labrax, U. cirrosa, D. dentex and E. marginatus specimens

Species	Parameters measured

	Protein (mg ml⁻¹)	pH	Conductivity (mS cm⁻¹)	Redox potential (mV)	Osmolality (mmol kg⁻¹)
Sparus aurata	0.88 ± 0.01	7.2	11.7 ± 0.6	191.9 ± 1.1	1,104 ± 4.51
Dicentrarchus labrax	1.45 ± 0.02	6.7	9.2 ± 0.5	156.5 ± 1.3	595 ± 1.76
Umbrina cirrosa	0.92 ± 0.01	7.2	7.6 ± 0.7	193.2 ± 0.9	1,010 ± 13.5
Dentex dentex	1.12 ± 0.01	7.1	9.4 ± 0.5	190.2 ± 1.2	934 ± 2.08
Epinephelus marginatus	0.90 ± 0.01	7.2	9.7 ± 0.4	178.2 ± 0.8	765 ± 2.51

Note: Data represent the value of a pool of 30 fish ± SEM of the technical replicates.

Table 2

Density measurements (g ml⁻¹) of skin mucus of S. aurata, D. labrax, U. cirrosa, D. dentex and E. marginatus specimens

Species	Density measurements (g ml⁻¹)

	12°C	17°C	22°C
Sparus aurata	1.028	1.027	1.026
Dicentrarchus labrax	1.014	1.013	1.012
Umbrina cirrosa	1.025	1.024	1.023
Dentex dentex	1.018	1.017	1.016
Epinephelus marginatus	1.024	1.023	1.021
Water	0.9995	0.9988	0.9978

Notes: Data represent the mean value of a pool of 30 fish. All standard deviations for technical replicates are ± 0.002.

First, we wanted to evaluate the viscosity of the skin mucus because this property is of major concern for the adhesion and entry of pathogens. Thus, viscosity showed a non-Newtonian behaviour (more intense in the samples with higher viscosity) in the skin mucus that is also described in the literature for seawater-reared Atlantic salmon (Salmo salar), brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss) and plaice (Pleuronectes platessa L.) while it was not clearly observed when Atlantic salmon and rainbow trout were reared in freshwater [35,37]. The shear rates used in this study were the same as utilized by Roberts and Powell [35]: 11.5, 23, 46 and 115 s⁻¹, which represent swimming velocities of 0.45, 0.91, 1.81 and 4.52 m s⁻¹, some of which have already been used in laboratory swimming trials for Atlantic salmon, brown trout, gilthead seabream and European sea bass [38–41]. In addition, shear rate around 450 s⁻¹ (corresponding to 17 m s⁻¹) was also used to simulate a faster swimming of fish. According to Roberts and Powell [35], when fish increases their swim speed, the mucin aggregates in skin mucus become broken and elongated, and line up with the streamlines, creating a slippage plane. In this way, flow resistance is reduced and viscosity becomes water-like. After, when shear stops, the viscosity of mucus recovers, restoring much of its original viscosity within seconds [42]. This behaviour of skin mucus helps fish locomotion by reducing fluid friction and enhancing movement through water [43,44].

The difference in the values of viscosity can be also related with other physico-chemical parameters with the aim to gain more understanding about this behaviour. Thus, if we pay attention to the amount of protein present in mucus, we can compare our extreme cases in D. labrax and S. aurata because the first has nearly double the amount of protein compared to the second. Density of the same samples can be included in the comparison. Mucus from D. labrax is the least dense and that from S. aurata the densest of those studied. In addition, although much care must be taken when correlating density with osmolality (the first one is mass per volume of solution and the second is number of solute particles per mass of solvent), if we take the approximation that, at the same temperature, for each molecule or ion present in the solution the volume only depends on its nature, osmolality values show that the number of particles (molecules or ions) present in D. labrax is around half of that present in S. aurata. These results show that mucins are the main solutes responsible for viscosity (it is well known that the contribution to viscosity is much higher for macromolecules than from small molecules) and also that the negative correlation is very good between mg of protein (per ml) and density as a consequence that small molecules are better packed than proteins in skin mucus.

In addition, for the whole set of samples, we find that the same conclusions can be obtained if we include D. dentex and E. marginatus. As an exception, one might think that U. cirrosa does not follow these conclusions. In fact, with an amount of protein very similar to S. aurata and E. marginatus, it shows a very similar viscosity to D. labrax and D. dentex, and with a slightly higher amount of protein shows a slightly higher density than E. marginatus. But the situation can be clarified if we realize that osmolality values show that there are more small particles in the same volume of mucus of U. cirrosa than in mucus of E. marginatus. This explains the values of density versus content in protein.

Regarding viscosity we can also explain the behaviour. If we compare S. aurata (low viscosity) and U. cirrosa (high viscosity), two mucus with similar content in protein and similar number of particles (similar osmolality), we find that the main difference appears in conductivity values (identical pH and redox potential). This difference in conductivity can be caused by a difference in the number of charged particles (ions) present in the fluid or by a difference in the motility of these particles. Thus, it is may be that the number of ions is higher in U. cirrosa, probably slightly affecting the conformation of proteins, making the solution more viscous and reducing the motility of the ions. These conclusions may also apply to the two mucus with lower viscosity (S. aurata and E. marginatus). The content in protein is similar but the number of small particles is lower in E. marginatus than in S. aurata, and the value of conductivity can be explained by the proportional reduction in the number of small charged particles.

Our results clarify findings from previous studies. For example, Roberts and Powell [35] observed a positive correlation between viscosity and osmolality, with lower viscosity in fish reared in freshwater than in seawater. Similarly, Antonova et al. [45] described clinical synthetic lung surfactants that produced an increase in the viscosity in response to increased salinity. But, as we have shown above, a higher value in osmolality does not necessarily cause a higher value in viscosity unless a certain change in the fluid structure is produced, for example, a change in protein conformation due to a variation of the ionic strength of the solution.

Some more comments can be done about the properties measured in this work. As expected, viscosity decreased with temperature whilst a decrease in mucus density was observed with increasing temperature in all species. The quantitative variations in mucus and water densities with temperature were essentially identical, which is an indication that no significant structural change was produced in the components of the skin mucus in the range of temperatures studied. For redox potential, which is a measure of the activity of the electrons and the tendency of the chemical species to accept them, values are positive and small and show no clear correlation with the protein content, indicating that proteins present in mucus do not have chemical groups which are easily reduced. To our knowledge, no studies have related these parameters with biological functions. Regarding the pH, some species can adopt different forms depending on the amount of protons present in the media and conductivity can be strongly affected by the presence of H⁺ or OH⁻. In addition, a practical implication of this parameter is the demonstration that bacterial attachment to fish surfaces was pH-dependent [46,47]. Balebona and coworkers [47] also observed a similar adhesion pattern in all the strains tested at alkaline pH, with the highest adhesion in the pH range of 8.2–8.5. However, at slightly acid or neutral pH values, bacterial adhesion to mucus varied between the different strains. As we can see from Table 1, differences in our samples due to this factor are likely to be negligible.

Osmolality may indicate great ion gradients in skin mucus. Ion gradients between the surrounding water and mucus would offer a reduced ion gradient to the plasma, thereby reducing the cost of ion transport [48]. Contrarily, Roberts and Powell [35] observed slight differences of osmolality between three seawater fish species, which may indicate small ion gradients. Taking into account that the seawater osmolality measured by us was 1,106 ± 3.2 mmol kg⁻¹, we could consider that skin mucus of S. aurata, U. cirrosa and D. dentex is iso-osmotic, while the skin mucus of D. labrax and E. marginatus could be categorized as hypo-osmotic to the surrounding water. Thus, in the case of iso-osmotic mucus the energy destined to ionic transport appears unimportant, compared to the situation in hyper-osmotic mucus [35].

Differences in mucin glycoproteins can occur in protein (simple and complex), carbohydrate, lipid and mineral content [43,49]. In our study, protein concentration was always higher than those found in skin mucus of salmonids [35]. Variations can be attributed to the interspecific differences, since all fish specimens used in the present work were reared in the same aquaria conditions. Interestingly, exposure of seabream specimens to heavy metals produced very little changes in the terminal carbohydrate profiles of mucus but the protein amount and diversity was clearly increased [50]. This fact suggests that physical and/or chemical properties of skin mucus might be affected by the environment.

In conclusion, we have measured the viscosity of skin mucus from five marine teleost species and some physico-chemical properties (pH, conductivity, redox potential, density, osmolality and density) to support and extend the viscosity results. For the samples studied, mucus viscosity showed a non-Newtonian behaviour with two different groups. Our results enlarge and help to clarify the few results shown in the literature, especially the correlation between osmolality or salinity and viscosity. Viscosity might be related to the ability of pathogens to penetrate and cross the mucus barrier but this has never been proven, and other physico-chemical parameters of skin mucus parameters may also play a significant role in pathogen adhesion and/or entry. It is plausible to propose that a high viscosity in the skin mucus should increase the barrier effect for pathogens due to their slower diffusion and therefore the higher time needed to penetrate and reach the epithelial surface. At the same time, a low viscosity could improve locomotion. The non-Newtonian behaviour of the skin mucus may represent a mechanism to find a compromise between the two requirements. Consequently, characterization of the relationship between these parameters and disease susceptibility, and thus improved understanding of the biology and function of the fish skin mucus barrier deserves further attention.

Footnotes

Acknowledgements

The financial support of the Spanish Ministerio de Economía y Competitividad under Grant no. AGL-2011-30381-C03-01 and of the Fundación Séneca de la Región de Murcia (Spain) (Grant no. 04538/GERM/06 within a Grupo de Excelencia de la Región de Murcia and Grant no. 19499-PI-14) is gratefully acknowledged. F.G.D.B. is grateful to Prof. García de la Torre for providing resources funded by grants from the Spanish Ministerio de Economía y Competitividad (project CTQ2012-33717) and Fundación Séneca (Grant no. 04531/GERM/06, within a Grupo de Excelencia de la Región de Murcia).

References

[1] Van der Marel

Caspari

Neuhaus

Meyer

Enss

Steinhagen

. Changes in skin mucus of common carp, Cyprinus carpio L., after exposure to water with a high bacterial load. J Fish Dis. 2010;33:431–439. doi:10.1111/j.1365-2761.2010.01140.x.

[2] Shephard

. Functions for fish mucus. Rev Fish Biol Fish. 1994;4:401–429. doi:10.1007/BF00042888.

[3] Spitzer

Koch

. Hagfish skin and slime glands. In: Jorgensen

Lomholt

Weber

Malte

, editors. The biology of hagfishes. London: Chapman Hall; 1998. p. 109–132.

[4] Subramanian

MacKinnon

Ross

. A comparative study on innate immune parameters in the epidermal mucus of various fish species. Comp Biochem Physiol B Biochem Mol Biol. 2007;148:256–263. doi:10.1016/j.cbpb.2007.06.003.

[5] Raj

Fournier

Rakus

Ronsmans

Ouyang

Michel

, et al. Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells. Vet Res. 2011;42:92. doi:10.1186/1297-9716-42-92.

[6] Esteban

. An overview of the immunological defenses in fish skin. ISRN Immunol. 2012;2012:853470. doi:10.5402/2012/853470.

[7] Khong

Kuah

Jaya-Ram

Shu-Chien

. Prolactin receptor mRNA is upregulated in discus fish (Symphysodon aequifasciata) skin during parental phase. Comp Biochem Physiol B Biochem Mol Biol. 2009;153:18–28. doi:10.1016/j.cbpb.2009.01.005.

[8] Lindenstrom

Buchmann

Secombes

. Gyrodactylus derjavini infection elicits IL-1β expression in rainbow trout skin. Fish Shellfish Immunol. 2003;15:107–115. doi:10.1016/S1050-4648(02)00142-0.

[9] Cole

Weis

Diamond

. Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem. 1997;272:12008–12013. doi:10.1074/jbc.272.18.12008.

10.

[10] Fernandes

JMO

Smith

. Partial purification of antibacterial proteinaceous factors from erythrocytes of Oncorhynchus mykiss . Fish Shellfish Immunol. 2004;16:1–9. doi:10.1016/S1050-4648(03)00027-5.

11.

[11] Guardiola

Cuesta

Arizcun

Meseguer

Esteban

. Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata). Fish Shellfish Immunol. 2014;36:545–551. doi:10.1016/j.fsi.2014.01.001.

12.

[12] Concha

Molina

Oyarzún

Villanueva

Amthauer

. Local expression of apolipoprotein A-I gene and a possible role for HDL in primary defence in the carp skin. Fish Shellfish Immunol. 2003;14:259–273. doi:10.1006/fsim.2002.0435.

13.

[13] Dalmo

Ingebrigtsen

Bogwald

. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). J Fish Dis. 1997;20:241–273. doi:10.1046/j.1365-2761.1997.00302.x.

14.

[14] Itami

. Defense mechanism of Ayu skin mucus. J Shimonoseki Univ Fish. 1993;42:1–71.

15.

[15] Tsutsui

Tasumi

Suetake

Suzuki

. Lectins homologous to those of monocotyledonous plants in the skin mucus and intestine of pufferfish, Fugu rubripes . J Biol Chem. 2003;278:20882–20889. doi:10.1074/jbc.M301038200.

16.

[16] Tsutsui

Tasumi

Suetake

Kikuchi

Suzuki

. Demonstration of the mucosal lectins in the epithelial cells of internal and external body surface tissues in pufferfish (Fugu rubripes). Dev Comp Immunol. 2005;29:243–253. doi:10.1016/j.dci.2004.06.005.

17.

[17] Aranishi

Mano

. Response of skin cathepsins to infection of Edwardsiella tarda in Japanese flounder. Fish Sci. 2000;66:169–170. doi:10.1046/j.1444-2906.2000.00026.x.

18.

[18] Hatten

Fredriksen

Hordvik

Endresen

. Presence of IgM in cutaneous mucus, but not in gut mucus of Atlantic salmon, Salmo salar. Serum IgM is rapidly degraded when added to gut mucus. Fish Shellfish Immunol. 2001;11:257–268. doi:10.1006/fsim.2000.0313.

19.

[19] Xu

Parra

Gómez

Salinas

Zhang

von Gersdorff Jørgensen

, et al. Teleost skin, an ancient mucosal surface that elicits gut-like immune responses. Proc Natl Acad Sci USA. 2013;110:13097–13102. doi:10.1073/pnas.1304319110.

20.

[20] LaFrentz

LaPatra

Jones

Congleton

Sun

Cain

. Characterization of serum and mucosal antibody responses and relative per cent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum . J Fish Dis. 2002;25:703–713. doi:10.1046/j.1365-2761.2002.00424.x.

21.

[21] Roussel

Delmotte

. The diversity of epithelial secreted mucins. Curr Org Chem. 2004;8:413–437. doi:10.2174/1385272043485846.

22.

[22] Thornton

Sheehan

. From mucins to mucus: Toward a more coherent understanding of this essential barrier. Proc Am Thorac Soc. 2004;1:54–61. doi:10.1513/pats.2306016.

23.

[23] Bansil

Turner

. Mucin structure, aggregation, physiological functions and biomedical applications. Curr Opin Colloid Interface Sci. 2006;11:164–170. doi:10.1016/j.cocis.2005.11.001.

24.

[24] Perez-Vilar

Hill

. The structure and assembly of secreted mucins. J Biol Chem. 1999;274:31751–31754. doi:10.1074/jbc.274.45.31751.

25.

[25] Nigam

Kumari

Mittal

. Comparative analysis of innate immune parameters of the skin mucous secretions from certain freshwater teleosts, inhabiting different ecological niches. Fish Physiol Biochem. 2012;38:1245–1256. doi:10.1007/s10695-012-9613-5.

26.

[26] McGuckin

Lindén

Sutton

Florin

. Mucin dynamics and enteric pathogens. Nat Rev Microbiol. 2011;9:265–278. doi:10.1038/nrmicro2538.

27.

[27] Yan

. A histochemical study on the snout tentacles and snout skin of bristlenose catfish Ancistrus triradiatus . J Fish Biol. 2009;75:845–861. doi:10.1111/j.1095-8649.2009.02326.x.

28.

[28] Andrianifahanana

Moniaux

Batra

. Regulation of mucin expression: Mechanistic aspects and implications for cancer and inflammatory diseases. Biochim Biophys Acta-Rev Cancer. 2006;1765:189–222. doi:10.1016/j.bbcan.2006.01.002.

29.

[29] Rose

Voynow

. Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol Rev. 2006;86:245–278. doi:10.1152/physrev.00010.2005.

30.

[30] Mittal

Ueda

Fujimori

Yamada

. Histochemical analysis of glycoproteins in the unicellular glands in the epidermis of an Indian freshwater fish Mastacembelus pancalus (Hamilton). Histochem J. 1994;26:666–677. doi:10.1007/BF00158292.

31.

[31] Kumari

Yashpal

Mittal

. Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae). Tissue Cell. 2009;41:271–280. doi:10.1016/j.tice.2008.12.006.

32.

[32] Bradford

. A rapid and sensitive method for quantification of microgram quantities of protein using the principle of protein dye binding. Anal Biochem. 1976;72:248–254. doi:10.1016/0003-2697(76)90527-3.

33.

[33] Ingram

. Substances involved in the natural resistance of fish to infection. J Fish Biol. 1980;16:23–60. doi:10.1111/j.1095-8649.1980.tb03685.x.

34.

[34] Guardiola

Cuesta

Abellán

Meseguer

Esteban

. Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish. Fish Shellfish Immunol. 2014;40:24–31. doi:10.1016/j.fsi.2014.06.018.

35.

[35] Roberts

Powell

. The viscosity and glycoprotein biochemistry of salmonid mucus varies with species, salinity and the presence of amoebic gill disease. J Comp Physiol B. 2005;175:1–11. doi:10.1007/s00360-004-0453-1.

36.

[36] Chiou

Wang

. Production and rheological characterization of the viscoelastic biopolymer produced by an eel epithelial cell line. Plant Pathol. 2000;23:551–556. doi:10.1007/s004499900161.

37.

[37] Lopez-Vidriero

Jones

Reid

. Analysis of skin mucus of plaice Pleuronectes platessa . J Comp Path. 1980;90:415–420. doi:10.1016/0021-9975(80)90011-0.

38.

[38] Okland

Finstad

McKinley

Thorstad

Booth

. Radio-transmitted electromyogram signals as indicators of physical activity in Atlantic salmon. J Fish Biol. 1997;51:476–488. doi:10.1111/j.1095-8649.1997.tb01505.x.

39.

[39] Thorstad

. Radio-transmitted electromyogram signals as indicators of swimming speed in lake trout and brown trout. J Fish Biol. 2000;57:547–561. doi:10.1006/jfbi.2000.1336.

40.

[40] Andrew

Noble

Kadri

Jewell

Huntingford

. The effect of demand feeding on swimming speed and feeding responses in Atlantic salmon Salmo salar L., gilthead sea bream Sparus aurata L. and European sea bass Dicentrarchus labrax L. in sea cages. Aquac Res. 2002;33:501–507. doi:10.1046/j.1365-2109.2002.00740.x.

41.

[41] Basaran

Ozbilgin

. Effect of lordosis on the swimming performance of juvenile sea bass (Dicentrarchus labrax L.). Aquac Res. 2007;38:870–876. doi:10.1111/j.1365-2109.2007.01741.x.

42.

[42] Cone

. Mucus. In: Ogra

Mestecky

Lamm

Strober

Bienestock

McGhee

, editors. Mucosal immunology. Academic Press; 1999. p. 43–64.

43.

[43] Lebedeva

. Skin and superficial mucus of fish: Biochemical structure and functional role. In: Saksena

, editor. Ichthyology recent research advances. Science Publishers; 1999. p. 177–193.

44.

[44] Rosen

Cornford

. Fluid friction of fish slimes. Nature. 1971;234:49–51. doi:10.1038/234049a0.

45.

[45] Antonova

Todorov

Exerowa

. Rheological behavior and parameters of the in vitro model of lung surfactant systems: The role of the main phospholipid component. Biorheology. 2003;40:531–543.

46.

[46] Gordon

Gerchakov

Udey

. The effect of polarization on the attachment of marine bacteria to copper and platinum surfaces. Can J Microbiol. 1981;27:698–703. doi:10.1139/m81-108.

47.

[47] Balebona

Moriñigo

Faris

Krovacek

Mhsson

Bordas

, et al. Influence of salinity and pH on the adhesion of pathogenic Vibrio strains to Sparus aurata skin mucus. Aquaculture. 1995;132:113–120. doi:10.1016/0044-8486(94)00376-Y.

48.

[48] Handy

. The ionic composition of rainbow trout body mucus. Comp Biochem Physiol Part A Physiol. 1989;93:571–575. doi:10.1016/0300-9629(89)90012-1.

49.

[49] Litt

. Comparative studies of mucus and mucin physicochemistry. In: Nugen

O’Conner

, editors. Ciba Foundation Symposium 109 – Mucus and mucosa. Pitman Publishing; 1984. p. 196–211.

50.

[50] Guardiola

Dioguardi

Parisi

Trapani

Meseguer

Cuesta

, et al. Evaluation of waterborne exposure to heavy metals in innate immune defences present on skin mucus of gilthead seabream (Sparus aurata). Fish Shellfish Immunol. 2015;45:112–123. doi:10.1016/j.fsi.2015.02.010.