Mar Biol (2010) 157:261–267 DOI 10.1007/s00227-009-1313-z
ORIGINAL PAPER
Measuring mucus thickness in reef corals using a technique devised for vertebrate applications Amita A. Jatkar · Barbara E. Brown · John C. Bythell · Reia Guppy · Nicholas J. Morris · JeVery P. Pearson
Received: 27 March 2009 / Accepted: 24 September 2009 / Published online: 11 October 2009 © Springer-Verlag 2009
Abstract A method previously used to measure thickness of the surface mucus layer (SML) of the mammalian gastrointestinal tract has been applied to the SML of reef corals. It involves manual measurement of mucus thickness using a micromanipulator and Wne glass needle (micropipette) and is non-destructive to the coral, meaning that repeated measurements can be taken. A measurable mucus layer was recorded in all cases in the study, which comprised 450 individual thickness measurements from four coral species. Mucus thickness ranged from 145 to 700 m. Thus, whatever dynamic processes control mucus synthesis, secretion to the tissue surface and subsequent release into the water column, a continuous mucosal barrier is maintained. A change in SML thickness was recorded as a response to aerial exposure during the natural tidal cycle and to solar exposure-induced bleaching, although the response due to bleaching varied between two studied species. The technique is rapid, cost-eVective and a simple means of assessing coral SML thickness, a variable that shows signiWcant variation in relation to environmental
Communicated by T. L. Goulet. A. A. Jatkar (&) · B. E. Brown · J. C. Bythell · R. Guppy School of Biology, Newcastle University, Newcastle upon Tyne NE1 7RU, UK e-mail:
[email protected] N. J. Morris School of Biomedical Sciences, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK J. P. Pearson Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK
conditions and is likely to be an important health indicator in these organisms.
Introduction Mucus secretory cells are abundant and widely distributed in both the epidermis and gastrodermis of healthy coral tissues, and mucus secretion forms an important part (20– 45%) of the energetic demands of reef-building corals and represents an even greater proportion of the nitrogen requirements (Brown and Bythell 2005). It was initially thought mainly to aid the functions of sediment removal (Hubbard and Pocock 1972) and ciliary feeding (Duerden 1906; Yonge 1930), but a variety of other roles have emerged, some of which are still poorly understood. One such major role of the surface mucus layer (SML) is protection against a range of hostile conditions including desiccation, microbial invasion, sedimentation and pollutants (Brown and Bythell 2005). An immediate increase in surface mucus secretion has been reported as a response to environmental insults such as presence of crude oil (Mitchell and Chet 1975; NeV and Anderson 1981), copper sulphate exposure (Mitchell and Chet 1975), decreased salinity and sediment overload (CoVroth 1985). An increased abundance and size of epidermal mucus secretory cells was seen in the reef coral Manicina areolata on long term exposure to crude oil (Peters 1981). Similar increased abundance of epidermal mucus-producing cells was reported in Goniastrea aspera during a natural bleaching event elicited by elevated sea surface temperature (Brown et al. 1995). Thus, these studies indicate a likely increase in mucus synthesis and secretion in response to environmental stress. However, further studies are needed to address the relative rates of synthesis, secretion to the tissue surface and release
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to the surrounding water column in relation to both the rate of metabolism and environmental stress responses (Brown and Bythell 2005). The SML possesses many beneWcial properties which aid in the protection of the underlying epithelia (Variyam 1995, 1996a, b, 2007; Belley et al. 1999; Moncada et al. 2005). For example, the surface of epithelial cells and the mucus gel in the human intestine have been found to contain similar ligands recognised by amoebic adherence glycoprotein. The adherence of amoebic trophozoites to the mucus gel, which eventually sloughs oV, thereby protects the epithelium from infection (Variyam 1995, 1996a, b, 2007; Belley et al. 1999; Moncada et al. 2005). In humans, the colon is densely colonised by anaerobic bacteria (1011–1012 cells ml¡1 of intestinal content), which occupy virtually every available niche in the outer mucus layer (Laux et al. 2005). This natural gut micro-Xora, together with the biophysical properties of the mucus layer itself, provide protection from the infective stages of pathogens and several mechanisms have been elucidated for this protective role. For example, the beneWcial bacteria of the gut micro-Xora have been shown to produce glycosidases and proteases that degrade the resistant lectin covering of Entameoba histolytica trophozoites, which renders them less capable of binding to epithelial cell surfaces (Variyam 2007). As well as direct competitive roles, pro-biotic bacteria such as Lactobacillus plantarum and L. rhamnosus have been shown to inhibit the in vitro adherence of Escherichia coli to the intestinal lining of human epithelial cells by stimulating the host cells to up-regulate the expression of MUC2 and MUC3 mucins (Mack et al. 1999). Thus, the mucus gel layer and its micro-Xora play a signiWcant role in limiting the adherence and penetration of pathogens. While relatively little is known about these processes in reef corals, the protective role of the SML has been widely hypothesised (Brown and Bythell 2005), and a reduction of antibacterial activity in the mucus and a shift in the microbial community to dominance by potentially harmful Vibrio species have recently been reported due to coral bleaching and disease (Ritchie 2006; Bourne et al. 2008). Thus, there is growing evidence suggesting that the SML promotes beneWcial microbial communities to develop by creating a favourable micro-environment inside the layer under healthy conditions. The eYciency of an SML as a protective physical barrier will depend on its thickness, composition, gel strength (Sellers et al. 1988), the structure of the layer and the dynamics of movement and release from the tissue surface. Of these factors, only mucus composition has been studied in any detail in corals (Ducklow and Mitchell 1979a, b; Daumas et al. 1981; CoVroth 1984, 1990; Meikle et al. 1988). However, even the chemical composition of coral mucus has remained questionable since various authors
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have used widely diVerent analytical techniques and sample collection methods, resulting in wide variation in its reported composition (Brown and Bythell 2005). Mucus composition and production rates will also likely vary depending on both zooxanthellae and host coral metabolic activity, although these eVects have scarcely been studied to date. Mucus composition has been reported for several coral species with characteristics being inXuenced by seawater depth, ageing and the level of contamination by surrounding water and particulate material (Ducklow and Mitchell 1979a, b; Daumas et al. 1981; CoVroth 1984, 1990; Meikle et al. 1988). The thickness and continuity of the mucus layer is routinely used as a measure of its protective capacity in the gastro-intestinal layer of humans and other mammalian models (Swidsinski et al. 2007; Phillipson et al. 2008; Strugala et al. 2008). Few analyses of mucus layer thickness have been attempted for reef corals, perhaps due to the diYculty of such measurements in the aquatic environment. Koren and Rosenberg (2006) estimated the SML thickness to be ca. 1 mm in Oculina patagonica, but this was based on a volumetric measurement of the total mucus released by a coral fragment under centrifugation and would thus include tissue stores released under stress so would not accurately determine the in vivo SML thickness. Similarly, Wild et al. (2005) equated volumes of mucus released following aerial exposure to a thickness of between 0.3 and 3.8 mm in Acropora millepora, but this would not account for the dynamics of secretion versus release from the tissue surface and would also not provide a valid measure of in vivo SML thickness. Since mucus is a hydrated gel, histological processing will typically result in signiWcant shrinkage if not total dissolution and loss of the SML. One possible exception is freeze-sectioning (e.g. Marshall and Clode 2004); however, the potential for mucus release as a stress response during the freezing process is a potential issue, and no attempt has so far been made to measure in vivo SML thickness using these techniques. Here, we have adapted a methodology that has been routinely used to measure the thickness of the mucus layers in the mammalian (rat) gastrointestinal tract (Atuma et al. 2001; Brownlee et al. 2003; Strugala et al. 2003) for use in an aquatic environment. This is a Wrst attempt to measure the thickness of SML in reef-building corals in vivo and to examine its responses under diVerent environmental conditions.
Materials and methods Experimental set-up Coral SML thickness measurements were carried out at the Phuket Marine Biological Centre, Thailand in February
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2007. Similar sized colonies (approximately 8 £ 5 cm) of diVerent coral species were collected by detaching the bases of the skeleton from the substrate with a hammer and chisel, without touching the live tissue as far as possible. Detached colonies were collected under water and were maintained in this condition during transfer to the laboratory. The colonies were maintained in a running seawater aquarium for up to 1 h prior to measurements being taken. During handling, care was taken to minimise handling, maintain ambient temperatures and avoid exposure to excessive sunlight, although the collected corals were intertidal and well adapted to stressful conditions including aerial exposure. Each colony was then transferred on to an inclined base plate in a measuring tank without exposure to air to perform mucus layer thickness measurements. Mucus gel measuring system Surface mucus layer thicknesses were measured using a glass micropipette held by a micromanipulator (Leitz, Wetzlar, Germany). The micromanipulator was connected to a digital micrometer. The tip diameter of the glass micropipette was formed at 1–2 m by pulling the glass tubing (1.2 mm OD and 0.6 mm ID; Rederick Haer, Brunswick, ME) using a pipette puller (pp-83; Narishinge ScientiWc Instrument Laboratories, Tokyo, Japan, Fig. 1). Under a binocular microscope, a translucent mucus gel layer was visible covering the coral surface. A drop of seawater containing carbon particles (puriWed activated charcoal particles approximately 10 m in diameter, Kebo Laboratories) was introduced using a dropper pipette to allow the interface between the mucus layer and the seawater to be easily distinguished. The micropipette was brought to this point perpendicular to the coral surface (estimated
Fig. 1 Mucus gel measuring system, C coral, D digital indicator, DM dissecting microscope, B base plate, M micromanipulator, MT measuring tank, P micropipette
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visually), and movement in the carbon particles due to an initial depression of the mucus layer surface prior to it being penetrated by the needle was used as an indicator that the outer surface of the mucus layer had been reached. The micrometer was then used to measure the distance between this point and the coral tissue surface, which was easily distinguished due to its natural pigmentation. The studied corals had a corallite (cup) diameter of between 4 and 17 mm, which is subdivided by vertical radiating septa. Typically, the application of the micropipette tip to the coral tissues resulted in a small localised retraction of the polyp. The advancement of the micropipette was stopped as soon as any localised retraction was observed or the tip was observed to contact the tissue surface. The coral surface is highly irregular and SML thickness varies signiWcantly over the tissue surface, with the area around the polyp mouth at the centre of the corallite having a thicker mucus layer than the tissues covering the tops of the septal ridges in between corallites. Hence in this study, a standardised location approximately mid-way between the ridge and deepest part of the corallite and in between two septa was chosen for all measurements. Estimation of error variance and potential bias Corals possess a variety of shapes ranging from smooth rounded to highly uneven branched forms. The mucus gel Xows and therefore acquires to some extent the shape of the underlying coral surface. One potential source of bias and error is the visual estimation of the perpendicular angle to the coral surface at the point of measurement. To assess this, a micropipette was inserted perpendicular to the coral surface (as estimated visually) and photographed from the side and printed so that the angular deviation from perpendicular could be manually assessed for that dimension using a protractor and set square. Absolute mean deviation from perpendicular (n = 15) was found to be 1.9 § 2.78° (§95% conWdence interval), which equates to an overestimation of the mucus thickness for a 400 m layer of just 0.12%. Thus, the potential bias and error due to visual estimation of the perpendicular angle to the coral surface is unlikely to be signiWcant. To determine the variance of SML thickness measurements across a colony, repeated measurements (n = 30) were taken from the standardised location of diVerent polyps across the coral surface of three diVerent colonies of Favites abdita. Power analysis was used to determine the optimum sample size using the pooled standard deviation obtained from these three colonies (SD = 151.7 m, Fig. 2). From this analysis, a sample size of n = 15 measurements per coral colony was used throughout the study, which resulted in a power of 0.41 to detect a diVerence of 100 m (or 19% of the mean thickness of 513 m) and a power of 0.94 to detect a diVerence of 200 m (39%).
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Fig. 2 Power curves for analysis of variance showing probability of detection of diVerences in mucus layer thickness for diVerent sample sizes based on an initial sample size of 30 for three separate colonies of Favites abdita. Pooled standard deviation SD = 151.73
Fig. 3 SML thickness measurements for three visibly healthy colonies of G. aspera and F. abdita (error bars 95% conWdence interval)
Examples of application of the method in vivo Four common coral species from the intertidal reef Xat in Phuket, Thailand were chosen for study: Goniastrea aspera, G. retiformis, Favites abdita, and Platygyra daedalea. These intertidal corals are aerially exposed at the study site during low spring tides, and solar bleaching is commonly observed following aerial exposure during periods of high irradiance (Brown et al. 2000, 2002). Visibly healthy colonies of G. aspera (n = 3) and F. abdita (n = 3) were collected on the same day to compare the SML thickness in the two species. The mean SML thickness was found to be 490 § 0.58 [95%CL] m and 496 § 0.59 [95%CL] m in G. aspera and F. abdita, respectively (Fig. 3). A nested ANOVA showed no signiWcant diVerence in SML thickness between coral colonies (F(1, 4) = 0.06, P = 0.5) or between the species (F(1, 4) = 0.06, P = 0.8). To assess the eVects of aerial exposure of corals during the natural tidal cycle, visibly healthy colonies of Goniastrea aspera (n = 3) were collected on four diVerent days at
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Fig. 4 SML thickness in G. aspera over diVerent days in a tidal cycle (error bars 95% conWdence interval). A-three colonies collected on the last day of a 5-day period when colonies had been aerially exposed daily; B, C and D-colonies collected on low tides in the tidal cycle when colonies were always submerged
the end of the spring tide sequence (Fig. 4). The colonies collected on the Wrst day had experienced the most extreme tidal conditions and had been repeatedly aerially exposed at low tide (in the early morning and mid afternoon) over a period of 5 days. The colonies collected over the subsequent 3 days from the same location had been continuously submerged throughout this period. There were highly signiWcant diVerences in SML thickness between collection days (nested ANOVA; F(3, 8) = 64.04, P < 0.001). The SML was 1.8-fold thicker in colonies collected on the Wrst day compared to the average SML of colonies collected subsequently. Colonies of G. retiformis (n = 3) and P. daedalea (n = 3) were selected to compare between areas of solar bleaching (visibly white tissues) that had developed on the west sides of colonies due to irradiance stress (Brown et al. 1994), and the rest of the colony which was normally pigmented. There was no signiWcant diVerence in SML thickness between coral colonies in P. daedalea in either the normally pigmented or bleached tissues (nested ANOVA; F(2, 3) = 1.12, P = 0.333), but the SML thickness on bleached tissue (315 m) was approximately half that of visibly coloured tissue (700 m; Fig. 5a; nested ANOVA; F(2, 3) = 21.72, P < 0.001). In contrast, G. retiformis showed no consistent pattern (Fig. 5b). Mucus thickness varied signiWcantly between coral colonies (nested ANOVA; F(2, 3) = 8.41, P < 0.001), and there was no signiWcant eVect of bleaching (nested ANOVA; F(2, 3) = 2.25, P = 0.088).
Discussion Obtaining realistic estimates of thickness of the delicate mucus gel layer in situ is diYcult, and the presence of the surrounding seawater makes it even more complicated in
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Fig. 5 a SML thickness in bleached and healthy tissues of three colonies of P. daedalea. b SML thickness in bleached and healthy tissues of G. retiformis colonies. (Error bars are 95% conWdence intervals)
corals. While the mucus layer can sometimes be preserved using histological methods (Ducklow and Mitchell 1979b; Johnston and Rohwer 2007), Wxation, dehydration and embedding will likely lead to shrinkage and deformation. Conventional techniques often result in complete dissolution and loss of the surface mucus layer (Allen and Pearson 2000). A rapid extrusion of mucus from mucus producing cells and a continuous mucus layer has been visualised under the light microscope as a response to low calcium/calcium free water in frozen sections of Galaxia fasicularis (Marshall and Clode 2004), and this method is promising since it requires no dehydration that will lead to shrinkage. However, it is unlikely that freeze sectioning will be generally useful for mucus layer studies in corals since the freezing process is time consuming and may itself alter the mucus secretion rates, which are well known to respond to such environmental insults (NeV and Anderson 1981; Peters 1981; CoVroth 1985; Brown et al. 1995). Thus, it is challenging to study and measure the SML in corals without disturbing the natural rate of secretion and turnover of the SML. Any technique involving Wxation is destructive and can only provide a snapshot of the SML thickness. This paper describes a novel method to measure the thickness of coral surface mucus and provides an opportunity to examine the dynamics of the layer over time and under diVerent conditions in vivo.
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The method showed a measurable layer of mucus in all of the 450 readings undertaken during the study, and mucus thickness ranged from 145 to 700 m. A similar SML thickness was detected in visibly healthy colonies of two species (G. aspera and F. abdita), and the method was successful in detecting the changes in the mucus layer as a result of stressful conditions. The thickness of the SML of G. aspera was increased (1.8-fold) due to periods of tidal aerial exposure compared with periods of continuous submergence. This increase in mucus thickness upon aerial exposure is presumably a response to mitigate the deleterious eVects of desiccation or harmful ultraviolet radiation (Drollet et al. 1997; Teai et al. 1998) or both. A signiWcant eVect of solar bleaching was observed in P. daedalea in which the SML was reduced to 50% in bleached tissues compared to visibly healthy polyps. In contrast, G. retiformis SML thickness was variable between colonies and showed no signiWcant diVerences due to bleaching. These diVerences may reXect diVerent levels of severity of the solar bleaching in the two study species rather than a general diVerence in the response due to bleaching. This is the Wrst time any method has revealed a decrease in surface mucus secretion due to bleaching, which has been long speculated and which is likely related to the decreasing densities of zooxanthellae and lower photosynthetic carbon production (Fitt et al. 2001; Douglas 2003; Brown and Bythell 2005). Thus, the technique is eYcient and sensitive enough to detect the eVects of environmental factors on the thickness of the coral SML. Microscopic observations during the study also re-emphasised the functional role of mucus in ciliary transport (Figs. 6, 7) as a mechanism for cleansing the coral surface
Fig. 6 G. aspera—Healthy colonies showing carbon particle aggregates (C), micropipette (M) and mucus string entangled with carbon particles (S)
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References
Fig. 7 G. retiformis—bleached/healthy colony (HP healthy polyp, BP bleached polyp, CD carbon particles deposited on the tissue)
of particulates. The continuous and rapid release of carbon particle-entangled mucus strings from the colony edges into the surrounding seawater, and continuous maintenance of a measurable mucus layer (>145 m) on the coral surface highlights the dynamic nature of the SML and supports the conclusion that the SML is continuously and rapidly renewed. The described method is non-destructive and provides a more realistic measure of mucus thickness in vivo than those that have been attempted so far using bulk mucus secretion calculations. This method also allows the study of the mucus layer in semi-natural conditions underwater. Stress eVects on corals during measurements were minimised by careful handling, short acclimation times and by keeping corals under observation for the minimum time possible. Although carbon particles were used to visualise the outer layer of the SML, the carbon particles are chemically inert and not dense enough to penetrate the mucus layer and so did not contact the coral tissues and could not therefore be sensed by the coral. Since the measurement of the SML in living corals has not been attempted in the past, the current measurements are unique in providing comparative SML thickness measurements for environmental (tidal exposure) and physiological (solar bleaching) eVects. The study shows that the method can be eVectively used on small-sized colonies freshly collected from the Weld and is sensitive enough to detect environmentally realistic changes in SML thickness. Acknowledgments This work was supported by a grant from the Leverhulme Trust (F/00125/S). We also gratefully acknowledge the support of the Phuket Marine Biological Centre management and staV for providing lab facilities during Weldwork.
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