Temporal dynamics of epidermal responses of guppies Poecilia reticulata to a sublethal range of waterborne zinc concentrations

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Journal of Fish Biology (2009) 75, 2642–2656 doi:10.1111/j.1095-8649.2009.02457.x, available online at www.interscience.wiley.com

Temporal dynamics of epidermal responses of guppies Poecilia reticulata to a sublethal range of waterborne zinc concentrations C. Gheorghiu*†, D. J. Marcogliese‡ and M. E. Scott* *Institute of Parasitology, Macdonald Campus of McGill University, 21,111 Lakeshore Road, Ste-Anne de Bellevue, QC, H9X 3V9 Canada and ‡Fluvial Ecosystem Research Section, Aquatic Ecosystem Protection Research Division, Water Science and Technology Directorate, Science and Technology Branch, Environment Canada, St Lawrence Centre, 105 McGill, 7th Floor, Montreal, QC, H2Y 2E7 Canada (Received 11 February 2009, Accepted 23 September 2009) This study assessed the histological changes in the epidermis of guppies Poecilia reticulata induced by waterborne zinc (Zn). Laboratory-reared P. reticulata fry were maintained individually in separate vessels containing artificial water (8 μg l−1 Zn) to which 0, 15, 30, 60 or 120 μg l−1 Zn was added. Their epidermal response to Zn was monitored regularly over 4 weeks. Compared with controls, mucus was rapidly released and mucous cell numbers decreased at all concentrations. Thereafter mucous release, epidermal thickness, numbers and size of mucous cells fluctuated at a rate that varied with Zn concentration, but fluctuations declined after day 18. Results clearly highlight the dynamic nature of the epidermal response to sublethal concentrations of waterborne Zn. In general, low concentrations of Zn induced a rapid response with reduced numbers and size of mucous cells and shift in mucin composition, and a subsequent thickening of the epidermis. Epidermal thickness and mucous cell area fluctuated over time but were normal after a month of exposure to low Zn concentrations. The number of mucous cells, however, remained low. Virtually all mucous cells from fish maintained in 15 and 60 μg l−1 Zn contained acidic mucins throughout the month, whereas fish maintained at 30 μg l−1 Zn responded by production of neutral mucins during the first 12 days followed by a mixture of neutral and acidic mucins. At 120 μg l−1 Zn, the most dramatic effects were the gradual but sustained decrease in numbers and area of mucous cells, and the shift to acidic mucins in these cells. Thus, as concentration of Zn increased, the epidermal responses indicated a disturbed host response (dramatic decline in mucous cell numbers, with mixed composition of mucins), which may have been less effective in preventing Zn uptake across the © 2009 Crown Copyright epithelium. Journal compilation © 2009 The Fisheries Society of the British Isles

Key words: acidic and neutral mucins; epidermal histology; epidermal thickness; fish epidermis; mucous cells; physiological adaptations.

INTRODUCTION The fish body surface is in intimate and continuous contact with the aquatic environment and therefore serves as the first line of defence against invading microorganisms and soluble contaminants (Iger et al., 1994; Cone, 1999). The fish epidermis consists †Author to whom correspondence should be addressed at present address: Department of Biology & Chemistry, Faculty of Science, Wilfrid Laurier University, 75 University Avenue West Waterloo, ON, N2L 3C5 Canada. Tel.: +1 519 884 0710, ext. 2347; email: cris [email protected]

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of epithelial and secretory cells and is covered by a layer containing mucus secreted by mucous (goblet) cells as well as the sloughed superficial layer of epidermal cells (Whitear, 1986). Both epithelial and secretory cells differentiate from a multipotent progenitor cell, and as new cells appear, the previously differentiated ones are gradually pushed towards the outer layer of the epidermis. The precursor mucous cell contains granules of neutral mucin glycoproteins exclusively (Sinha & Chakravorty, 1982). Under normal conditions, some of the neutral mucopolysaccharide granules are transformed into acidic mucopolysaccharide granules and new acid mucopolysaccharides are synthesized within the cell. In mature mucous cells, these acidic and neutral polysaccharide granules fuse forming a complex mixture of both acidic and neutral mucins (Sinha & Chakravorty, 1982) that form a protective barrier when released onto the surface of the fish (Whitear, 1986; Shephard, 1994). Epidermal exposure to stressors induces release of mucus and differentiation and migration of new mucous cells to the surface of the epidermis. As a metabolically active tissue, spatial and temporal changes occur in epithelium thickness, the density of mucous cells and the production and composition of mucus, depending on location on the body surface, fish species, age, state of health and nutritional status, and on the quality of the environment (Roberts & Bullock, 1980). Among the many environmental stresses that fishes face, waterborne contaminants including metals such as zinc (Zn) are of great concern. Zn is an indispensable micronutrient used as a growth-enhancing nutritional supplement in aquaculture (K¨ock & Bucher, 1997), but when its concentration exceeds that defined by water quality guidelines (30 μg l−1 Zn), Zn is considered as a pollutant (Canadian Council of Ministers of the Environment, 2005). In response to elevated waterborne Zn, the fish epidermis releases mucus (Iger et al., 1994; Shephard, 1994; Khunyakari et al., 2001) that binds Zn, thus preventing Zn absorption across the skin (Handy et al., 1989; Shephard, 1994). Acidic mucins are better at binding Zn than neutral mucins (N´ı Sh´uilleabh´ain et al., 2006), and thus up-regulation of biosynthesis and biotransformation of acidic mucins is a predicted consequence of Zn exposure. These epidermal processes presumably respond in a Zn concentration-dependent manner. Although continuing exposure to Zn might be expected to induce continual production of new mucous cells with sustained production and discharge of acidic mucins, physiological acclimation (Bradley et al., 1985) and increased tolerance (McGeer et al., 2000) to Zn have been reported in Oncorhynchus mykiss (Walbaum). In order to confer protection against contaminants and ectopathogens with which the skin is in intimate and continuous contact, the fish epidermis responds dynamically with changes in thickness and in the number, size and mucin composition of mucous cells. Moreover, as one of the most mobile body parts, the caudal peduncle is the first site of colonization for many invading pathogens (e.g. Gyrodactylus turnbulli ; Harris, 1988). Therefore, it is appropriate and important to understand the temporal dynamics of the epidermal response of the caudal peduncle to Zn before attempting to understand the response to combined stresses of Zn and infection. The present study was designed to characterize the response of guppy Poecilia reticulata Peters epidermis of the caudal peduncle (epidermal thickness, number, size and location of mucus cells, mucin composition and mucous production) to a range of sublethal concentrations of waterborne Zn over a period of 1 month. Poecilia reticulata was chosen as a laboratory model because they are easy to maintain and breed under laboratory conditions and because they can handle very high © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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concentrations of zinc (15-fold normal body concentration) without endangering their survival (Widianarko et al., 2000, 2001).

MATERIALS AND METHODS GENERAL METHODS The experiments were undertaken with fry of 0·5–1·0 cm standard body length (LS ), bred in the laboratory from a strain of feeder P. reticulata purchased from a local pet store. Each individual fry was maintained in a separate rectangular plastic container with 200 ml waterborne Zn solution at 25◦ C with 16L:8D cycle. Fish were fed a Nutrafin Max Complete Flake diet once a day. All procedures were approved by McGill University Animal Care Committee, in accordance with guidelines of the Canadian Council on Animal Care (2005). Artificial fresh water (AFW) was used for all experiments and was prepared according to Singh & Srinivastav (1993). Briefly, the AFW was made by adding different salts (0·123 g NaCl, 0·065 g Na2 SO4 , 0·004 g KCl, 0·117 g CaCl2 and 0·04 g MgCl2 per litre deionized water), and adjusted to pH 7·6 with NaHCO3 (Fisher Scientific, Montreal, Canada; www.fishersci.ca) to ion-free water. Sublethal concentrations for experimental waterborne Zn solutions were chosen relative to 30 μg l−1 Zn, the maximal admissible limit for aquatic life (Canadian Council of Ministers of the Environment, 2005). Zn solutions were prepared (Gheorgiu et al., 2006; correct spelling Gheorghiu) by adding 0, 15, 30, 60 or 120 μg l−1 Zn (as ZnCl2 with 98% purity; Anachemia Canada Inc., www.anachemia.com) to AFW. As previously shown (Gheorghiu et al., 2006), when a fish was kept for 2 days in the prepared AFW with no Zn added (control), the measured Zn concentration was c. 8 μg Zn l−1 . In addition, the same concentration of Zn was measured in the breeding tanks to which the fry had been exposed throughout life. Therefore, the appropriate control for the experiments was chosen to be c. 8 μg Zn l−1 instead of 0 μg Zn l−1 (Gheorghiu et al., 2006). According to previous results (Gheorghiu et al., 2006), relatively constant Zn concentrations can be maintained by complete replacement of Zn solutions every 2 days, and this procedure was followed for the present study. For convenience, results will be presented with respect to the nominal concentrations of Zn in the experimental solution (0, 15, 30, 60 or 120 μg l−1 Zn). Fish kept in a nominal waterborne Zn concentration of 0 μg l−1 were considered the control group. EXPERIMENT 1: EPITHELIAL RESPONSE TO ZN EXPOSURE The temporal changes induced by Zn were recorded on P. reticulata fry maintained throughout the experiment in individual containers. Each fish was exposed throughout the experiment to one of the five Zn solutions (nominal concentrations of 0, 15, 30, 60 or 120 μg l−1 Zn). Three fry per concentration were sampled at various intervals (0, 3, 6, 9, 12, 15, 18, 21 and 28 days) after Zn exposure began. The fry were killed in 0·03% tricaine methanesulphonate (Finquel MS-222, Argent Chemical Laboratories; www.argent-labs.com) buffered to a neutral pH with NaHCO2 , fixed for 24 h in Bouin’s solution, washed two times and then stored in 70% ethanol. Paraffin crosssections (5 μm) of the caudal peduncle were cut and stained using either haematoxylin and eosin (HE) for overall structure, or combined periodic acid-Schiff with alcian blue 2% (PAB) to differentiate between neutral polysaccharides and acidic mucopolysaccharides contained in the mucous cell (Tibbetts, 1997). When stained with PAB, the large swollen mature mucous cells were (1) purple if they contained both neutral and acidic mucins, (2) blue if they contained acidic mucins or (3) magenta if they contained neutral mucins (Bancroft & Stevens, 1982). The sections were examined using a compound microscope at ×425 magnification. Epidermal thickness, number of epidermal cell layers, number of mature mucous cells, their size, location and mucin composition were recorded from four lateral fields of view of 0·24 mm2 each, randomly chosen in each of three serial crosssections of the caudal peduncle per fish. Examples of histological characteristics are shown in Fig. 1. © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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Fig. 1. Histological images of epidermis of caudal peduncle of Poecilia reticulata fry (periodic acid-Schiff with alcian blue 2% staining, ×425). (a) Fish not exposed to Zn; mucous cells with a mixture of neutral and acidic mucins–[MC(m)]. (b) Fish exposed to 15 μg l−1 Zn for 3 days; mucous cells contained only acidic mucins [MC(a)]. (c) Fish exposed to 30 μg l−1 Zn for 6 days; mucous cells contained only neutral mucins [MC(n)]. ET, thickness of epidermis; EC, epithelial cells; S, fish scale.

E X P E R I M E N T 2 : M U C O U S P RO D U C T I O N I N R E S P O N S E TO ZN EXPOSURE As above, a total of 27 P. reticulata fry per concentration were isolated in individual containers and randomly assigned to one of four Zn solutions (15, 30, 60 or 120 μg l−1 Zn). At each of the time intervals mentioned above (see section Experiment 1), three of the fish per concentration were anaesthetized for a maximum of 5 min in 50 ml of 0·02% MS-222. Mucous discharge over the entire surface of the fish was recorded using a stereomicroscope with cold light and was assigned a score between 1 (minimal) and 5 (maximal) based on the percentage of the body surface covered and the thickness of grossly visible mucus. S TAT I S T I C A L A N A LY S I S The temporal effect of waterborne Zn on the guppy epidermis was assessed using χ 2 for categorical variables (mucous production scores, mucin composition and mucous cell location). Continuous histological variables were analysed by two-way ANOVA using PROC MIXED with a nested model that controlled for degrees of freedom, and post hoc contrasts across time within each Zn concentration using LSMEANS with the SLICE option. Linear regression analysis was used to examine the relationship between mean epidermal thickness © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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and mean number of cell layers per fish. The mean ± s.e. is reported. Analyses were performed using SAS version 9.1 software (www.sas.com). The level of significance was established at P < 0·05; statistics are reported only for significant effects.

RESULTS E P I D E R M A L T H I C K N E S S A N D C E L L L AY E R S

Nested ANOVA revealed that epidermal thickness (Fig. 2) was significantly affected by main effects of Zn concentration and time, as well as their interaction (Table I). Epidermal thickness [Fig. 2(a)] remained unchanged over time in the control group. Over the first 6 days, exposure to waterborne Zn did not influence epidermal thickness, but thereafter, the dynamic response of the epidermis was a function of Zn concentration. Exposure to 15 and 30 μg l−1 Zn induced fluctuations in epidermal thickness above initial values [Fig. 2(b), days 9 to 12; Fig. 2(c), days 9 to 15]. This pattern was repeated at 15 μg l−1 Zn but not at 30 μg l−1 Zn. In contrast, at higher concentrations (60 and 120 μg l−1 Zn), the epidermal thickness declined significantly on day 9 [Fig. 2(d), (e)], then increased significantly above initial values [Fig. 2(d), day 12; Fig. 2(e), days 12 to 15], then declined to [Fig. 2(d), day 15 to 18] or below [Fig 2(e), day 18 to 28] initial values. Thus, exposure to Zn induced fluctuations in epidermal thickness, with an overall decline in epidermal thickness as Zn concentration increased. The mean ± s.e. number of epithelial cell layers in the control group remained at 2·04 ± 0·04 over the course of the study. In response to Zn exposure, the number of cell layers followed the same pattern, and regression analysis revealed a positive relationship between mean epidermal thickness and mean number of epidermal cell layers per fish (P < 0·001). Table I. Two-way ANOVA results: main effects and the interactions between Zn concentration and duration of exposure for each variable measured in epidermis of Poecilia reticulata Effects Zn × duration of exposure

Variable

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Duration of exposure

Mucous production (ANOVA) Skin width (ANOVA)

F3,98 = 17·04 (P < 0·001) F3,1260 = 10·42 (P < 0·001) F3,1260 = 3·78 (P < 0·05) F3,1260 = 5·38 (P = 0·001) F3,2194 = 35·85 (P < 0·001) F3,294 = 18·30 (P < 0·001)

F7,98 = 64·03 F21,98 = 3·20 (P < 0·001) (P < 0·001) F8,1260 = 21·52 F24,1260 = 6·81 (P < 0·001) (P < 0·001) F8,1260 = 17·08 F24,1260 = 9·43 (P < 0·001) (P < 0·001) F8,1260 = 22·47 F24,1260 = 3·92 (P < 0·001) (P < 0·001) F8,2194 = 67·93 F24,2194 = 11·22 (P < 0·001) (P < 0·001) F8,294 = 5·14 F24,294 = 3·67 (P < 0·001) (P < 0·001) χ 2 = 1799·30 (P < 0·001)

Epithelial cell layers (ANOVA) Mucous cell number (ANOVA) Mucous cell area (ANOVA) Mucous cell location (ANOVA) Mucous cell type (χ 2 )

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Fig. 3. Temporal dynamics of mean ± s.e. mucous cell number in response to Zn: (a) 0, (b) 15, (c) 30, (d) 60 and (e) 120 μg l−1 Zn. Main effects: Zn, P < 0·001; time, P < 0·001; Zn × time, P < 0·001. Different lower case letters indicate significant differences for time effects.

MUCOUS RESPONSE

Control fish had 3·15 ± 0·40 mature mucous cells per 0·24 mm2 [Fig. 3(a)]. The mean ± s.e. area of these cells was 15·17 ± 0·11 μm2 , and 95% (95% CL = 91–98) were located within the external cell layer. These variables remained unchanged over time [Figs 3(a) and 4(a)]. Exposure to Zn induced a rapid decline in the number (Fig. 3) and size (Fig. 4) of mature mucous cells within 3 days, evident at all Zn concentrations, but especially so at 15, 30 and 60 μg l−1 Zn for cell numbers [Fig. 3(b) to (d)] and at 15 and 60 μg l−1 Zn for cell area [Fig. 4(b), (d)]. As with epidermal thickness, mucous cell numbers and size generally declined and became more © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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variable over time. Numbers returned to control values by day 15 in fish exposed to 15 and 60 μg l−1 Zn, but remained somewhat elevated at 30 μg l−1 Zn, and cell numbers remained depressed at 120 μg l−1 Zn throughout the experiment. Mucous cell area fluctuated and transiently returned to normal at the 15 μg l−1 Zn exposure [Fig. 4(b)] but at concentrations of ≥ 30 μg l−1 Zn, the cells remained small and were less than half the size of cells in unexposed fish after 28 days [Fig. 4(c) to (e)]. All mature mucous cells were located in the outer layer of the epidermis, regardless of Zn concentration or duration of exposure. The only exception was for fry exposed to 120 μg l−1 Zn where mean ± s.e., 19·1% ± 2·4 of mucous cells were localized in the inner layers. As expected, mature mucous cells contained a mixture of acidic and neutral mucins in fish not exposed to Zn [Fig. 5(a)]. Exposure to 15 μg l−1 Zn for 3 days induced an almost complete shift to acidic mucins that persisted until day 28 [Figs 1(b) and 5(b)]. An even more dramatic mucin response was seen in fish exposed to 60 μg l−1 Zn where all mucous cells had only acidic mucins from days 3 to 28 [Fig. 5(d)]. In contrast, at 30 and 120 μg l−1 Zn, mucin composition gradually shifted over the experiment, but in different ways. At 30 μg l−1 Zn, mucins were predominantly neutral between days 3 and 12 [Fig. 5(c)] and then predominantly mixed from days 15 to 28 [Fig. 5(c)]. At 120 μg l−1 Zn, mucins remained mixed during the first 9 days, but acidic mucins began to predominate after more prolonged Zn exposure [Fig. 5(e)]. Analysis of the semi-quantitative scores on visible mucus on the skin surface revealed that mucous production was significantly affected by Zn concentration (P < 0·001), by duration of exposure (P < 0·001) and by the interaction between these two factors (P = 0·001). Mucous scores increased then declined repeatedly over the first 18 days of Zn exposure (Fig. 6). The highest scores were recorded in fish kept in 15 and 30 μg l−1 Zn [Fig. 6(a), (b)], which showed very similar patterns. Intriguingly, data from fish exposed to 60 μg l−1 Zn indicates a damping of mucous release between days 6 and 12 [Fig. 6(c)] whereas mucous discharge on fish exposed to 120 μg l−1 Zn remained elevated throughout the first 9 days [Fig. 6(d)]. From day 18 until the end of the experiment, little mucus was visible regardless of Zn concentration (Fig. 6).

DISCUSSION Results of this study clearly highlight the dynamic nature of the epidermal response to sublethal concentrations of waterborne Zn. The first hypothesis was that exposure to Zn would induce an immediate release of mucus (Iger et al., 1994; Khunyakari et al., 2001), leading to a reduction in the number of mucous cells (Iger et al., 1988, 1994; Benedetti et al., 1989; Iger & Wendelaar Bonga, 1993). This was observed in all fish exposed to Zn, regardless of concentration. The second hypothesis was that hyperplasia induced by Zn (Iger et al., 1994) would lead to thickening of the epidermis, which in turn would be associated with an increase in the number of cell layers. Increased thickening of the epidermis was observed in response to lower Zn concentrations whereas at higher concentrations, the generalized response to Zn was a thinning of the epidermis. Finally, it had been predicted that higher concentrations of Zn and prolonged exposure to Zn would lead to increased proliferation of precursor mucous cells resulting in more cells of smaller size containing acidic mucins © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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Fig. 4. Temporal dynamics of mucous cell area of Poecilia reticulata in response to Zn: (a) 0, (b) 15, (c) 30, (d) 60 and (e) 120 μg l−1 Zn. Main effects: Zn, P < 0·001; time, P < 0·001; Zn × time, P < 0·001. Different lower case letters indicate significant differences for time effects.

© 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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Fig. 5. Frequency distribution of proportion of mucous cells with mixture of neutral and acidic mucins ( ), acidic mucins ( ) or neutral mucins ( ) in Poecilia reticulata fry exposed to different concentrations of waterborne Zn: (a) 0, (b) 15, (c) 30, (d) 60 and (e) 120 μg l−1 Zn. P < 0·001. © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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Fig. 6. Mean ± s.e. mucous production scores of Poecilia reticulata exposed to different concentrations of waterborne Zn: (a) 15, (b) 30, (c) 60 and (d) 120 μg l−1 Zn. Scores range from 1 (low) to 5 (high). Main effects: Zn, P < 0·001; time, P < 0·001; Zn × time, P < 0·001. Different lower case letters indicate significant differences for time effects.

(N´ı Sh´uilleabh´ain et al., 2006). Mucous cell numbers decreased, so the expected proliferation did not occur. In most cases after prolonged exposure at the higher concentrations, small cells (those < 10 μm2 ) containing acidic mucins were recorded. In discussing the results, three questions are addressed. Do current data show patterns similar to those reported in the literature? Are the responses likely to prevent entry of Zn across the skin? Is there evidence of acclimation of the fish to continued Zn exposure? D O C U R R E N T D ATA S H O W PAT T E R N S S I M I L A R T O T H O S E R E P O RT E D I N T H E L I T E R AT U R E ?

Data on the response of P. reticulata skin to waterborne Zn obtained from this study are broadly consistent with reports on gill (Bradley et al., 1985; Handy et al., 1989; McGeer et al., 2000), intestine (Sinha & Chakravorty, 1982) and skin (Sauer & Watabe, 1984; Iger et al., 1988; Nakano et al., 1992) epithelial responses to Zn and other metals, but indicate that the dynamics of these responses are much more complex than is apparent in the literature. Whitear (1986) reported that healthy P. reticualta have approximately seven mucous cells per mm length of epidermis, a value very similar to the observed 3·14 mucous cells per 0·49 mm of epidermis length. The acute response to 15 and 30 μg l−1 Zn, measured on day 3, involved a rapid elevation in mucous release on the skin surface, as evidenced by semi-quantitative mucous scores. This release of mucus presumably provided a first level of protection by trapping excess Zn and preventing its entry into underlying tissues (Shephard, 1994). A concurrent decrease by >50% in mucous cell numbers, as reported in O. mykiss (Iger et al., 1994) and brown bullhead Ictalurus nebulosus © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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(LeSueur) (Benedetti et al., 1989) was also observed. Between day 6 and 18, mucous release began to fluctuate as did epidermal thickness and mucous cell numbers. At higher concentrations of 60 and 120 μg l−1 Zn, fluctuations were not as marked. Although the observed fluctuations could reflect small sample size, the consistency in responses among fish at each time point within each concentration indicates an underlying biological explanation. The fluctuations could be due to concentrationdependent changes in the time delay between differentiation of multipotent progenitor cells induced by the sloughing of epidermal cells and the migration of the new cells to the surface of the epidermis. Similar fluctuations in epidermal thickness have been recorded in O. mykiss exposed to River Rhine water containing a mixture of pollutants including 30 μg l−1 Zn (Iger et al., 1994). A R E T H E E P I D E R M A L R E S P O N S E S L I K E LY T O P R E V E N T E N T RY O F Z N A C R O S S T H E S K I N ?

The interpretation of whether the observed tissue responses were helpful in preventing uptake of Zn across the skin epidermis is based on the current understanding of the role of mucus and mucins in binding and trapping Zn. Mucus is a complex mixture containing a variety of mucin glycoproteins as well as sloughed cells and other molecules (Whitear, 1986; Shephard, 1994). As expected (Sinha & Chakrovorty, 1982), mature cells on the outer layer of the epidermis contained a mixture of neutral and acidic mucins on day 0. By day 3, these mucins had been released onto the skin surface and the mucin content of the remaining or newly generated mucous cells had shifted dramatically. Virtually all mucous cells in fish exposed to 15 and 60 μg l−1 Zn contained only acidic mucins, indicating an immediate adaptive response in these fish. Acidic mucins are reportedly protective against Zn (Handy et al., 1989; Shephard, 1994) and the Golgi cisternae in mucous cells switch to producing strongly acidic mucins in response to a variety of stresses (Triebskorn et al., 1998). What was surprising, however, was the response of fish exposed to 30 and 120 μg l−1 Zn. First, it is unclear why exposure to 30 μg l−1 Zn would result in production of predominantly neutral mucins over the first 12 days of Zn exposure. Neutral mucins are the first mucins formed in precursor mucous cells (Sinha & Chakravorty, 1982). Perhaps, 30 μg l−1 Zn induced a more immediate differentiation of mucous cells. This is supported by the observation of internally localized mucous cells on day 3. A similar response was recorded in the epidermis of mummichog Fundulus heteroclitus (L.) exposed to contaminated sediment (M´ezin & Hale, 2000). The patterns of mucous cell numbers and mucous production, however, were quite similar between fish exposed to 15 and 30 μg l−1 Zn, so there is only limited evidence that the response to 30 μg l−1 Zn involved more rapid production of mucous cells. Of even more interest was the extremely gradual shift in mucin composition in fish exposed to 120 μg l−1 Zn. These fish continued to produce mixed acidic and neutral mucins during the first 15 days, and only at day 12 did evidence of a bias towards acidic mucins begin to appear. This delay in production of acidic mucins was not the result of a generalized lack of response of mucous cells, because mucous production scores remained elevated over the first 9 days of Zn exposure. Sustained synthesis and discharge is an energy-demanding process (Shephard, 1994) and conversion of neutral to acidic mucins as well as synthesis of more acidic mucins may also be energetically costly. When demands for mucous production are high (as © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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at 120 μg l−1 Zn), an energetic trade-off may occur between up-regulating mucous production and up-regulating the synthesis of acidic mucins. This hypothesis is consistent with the elevated mucous production and replacement of depleted mucous cells, but the absence of a shift to acidic mucins, that was observed in fish exposed to 120 μg l−1 Zn during the first 12–15 days. In general, it appears that fish exposed to 15 μg l−1 Zn were best able to handle waterborne Zn. Despite the initial drop in number and size of mucous cells, mucous cell area had increased to normal values on day 28 and the cells continually produced the protective acidic mucins. Fish exposed to 60 μg l−1 Zn used a different strategy in response to Zn exposure. They were quite successful in restoring mucous cell numbers to normal, and producing acidic mucins, but these cells remained very small. In contrast, it appears that the early responses of fish exposed to 30 and 120 μg l−1 Zn were less adaptive, either because of the profile of mucins produced (30 μg l−1 Zn) or because the numbers and size of mucous cells remained low, and the shift to acidic mucin production was delayed (120 μg l−1 Zn). I S T H E R E E V I D E N C E O F A C C L I M AT I O N O F T H E F I S H T O CONTINUED ZN EXPOSURE?

Although acclimation to metals has been well studied in fishes, to date the process seem to be more complex than initially considered. The survival of aquatic organisms in Zn polluted waters depends on the homeostatic control of both mechanisms of the absorption of Zn (mainly acquired through the gills and the gut) and mechanisms for limiting its assimilation and toxicity. After absorption of metals, the organism must either eliminate metals through excretion or sequester them to prevent toxicity (Sauer & Watabe, 1984). Fish surviving chronic, sublethal exposure to Zn (and other metals) are able to correct the ionic disturbance and to acquire physiological acclimation. After a short period of physical damage and disruption of physiological homeostasis, a recovery period follows, when tissue repair (especially studied in gills) begins and synthesis of MTs is upregulated (especially studied in the liver), thus re-establishing a homeostatic equilibrium with increased tolerance to the metal (McGeer et al., 2000). Current results indicated that epidermis from the caudal peduncle of P. reticulata responded dynamically to Zn and that the response was concentration-dependent. This is believed to be the first study to consider whether acclimation of the epidermis to Zn may explain concentration-dependent fluctuations in the fish epidermis. Disruption of the skin epidermis was evident on day 3 with the dramatic drop in number of mucous cells, followed by epidermal thickening at lower Zn concentrations, but thinning of the epidermis at the higher Zn concentrations over the next 6 days. Evidence of a tissue repair phase was seen in the fluctuating thickness of the epidermis, numbers of mucous cells and scores of mucous production over the first 18 days of Zn exposure. During the last 10 days of the study, fluctuations in epidermal variables had dampened, and both sustained low level of mucous production and low numbers of mucous cell regardless of Zn concentration were observed. This can be interpreted as either acclimation to Zn or perhaps an exhaustion of the epidermal tissue due to inability of sustained differentiation of progenitor cells (M´ezin & Hale, 2000). By day 28, mucous cells in fish maintained at 15 and 30 μg l−1 Zn had increased to normal size, and many of the cells contained the normal mixture of acidic and neutral mucins, suggesting possible acclimation. In © 2009 Crown Copyright Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2642–2656

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contrast, mucous cells in fish exposed for a month to higher concentrations of Zn remained at less than half the size of normal cells and contained only acidic mucins. This latter response may represent exhaustion or a possible chronic response, but confirmation will require further research. Although mucous is critically important in protecting the fish epidermis against entry of waterborne Zn, it is important to note that fishes also use the epidermis and scales as a storage tissue for Zn (Sauer & Watabe, 1984, 1989; Nakano et al., 1992), further preventing its entry and accumulation in other fish tissues. Thus sloughing of mucous and epidermal cells also serves as a mechanism to remove any Zn that may have been absorbed into these cells (Shephard, 1994). Both mucous release onto the surface and sloughing of cells may contribute to the high tolerance of P. reticulata to Zn (Widianarko et al., 2000, 2001). The positive correlation between tissue concentration of Zn and concentration of Zn in the sediment of urban streams from which P. reticulata had been collected (Widianarko et al., 2001), and the ability of P. reticulata to survive with tissue concentrations of Zn that were 15-fold higher than normal (Widianarko et al., 2000) indicate that additional physiological adaptations, probably including elevated MT levels, are also involved. Funding for this research was provided by the Natural Sciences and Engineering Research Council, Canada (NSERC 3585) together with the St Lawrence Action Plan (Environment Canada). The first author also acknowledges an NSERC Postgraduate Scholarship. Research at the Institute of Parasitology is supported by a Regroupement Strat´egique from FQRNT (a provincial funding agency). M. Assad and his team of technicians from the Centre for Bone and Periodontal Research (McGill University) processed histological samples.

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Electronic References Canadian Council on Animal Care (2005). Guidelines on: The Care and Use of Fish in Research, Teaching and Testing. Available at http://www.ccac.ca/en/CCAC Programs/ Guidelines Policies/GDLINES/Fish/Fish%20Guidelines%20English.pdf Canadian Council of Ministers of the Environment (2005). Canadian Water Quality Guidelines (CWQG) for the Protection of Aquatic Life. Available at http://www.ccme.ca/assets/pdf/ wqg aql summary table.pdf

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