Low pH modify cytokeratins (AE3 and HMW) localization pattern.
Both AQP3 and iNOS expression are induced by low pH in L. italicus larval skin.
Low pH activates a network of responses closely intertwined, in the larval skin.
Mounting evidence suggests that amphibians are globally and currently the most threatened group of vertebrates and different causes might be responsible for this phenomenon. Acidification of water bodies is a global environmental issue that has been proposed as a possible cause for amphibian populations decline. Indeed, it has been widely demonstrated that low pH may exert harmful effects on amphibians, either directly or by increasing the adverse effects of other stressors. Surprisingly only few studies documented the response of amphibian integument to acidic pH conditions and no data are available on the effects of a non-lethal level of pH onto the amphibian larval epidermis. The present study showed that acidic pH (4.5) condition has severe effects on the epidermis of the Italian newt (Lissotriton italicus, formerly Triturus italicus) inducing both morphological and functional alterations. The increase of mucus is the first evident effect of acid injury, followed by the flattening of the epithelium and the appearance of a keratinized shedding layer. The immunolabeling of cytokeratins substantially changes acquiring an adult-like pattern. Also aquaporin 3 and iNOS expression modify their distribution according to a change of the histological features of the epidermis. These results clearly indicate that a short-term exposure to a sub-lethal pH disrupts the epidermis morphology and function in L. italicus larvae. Since the skin exerts a prominent role in both respiration and osmoregulation, the described alterations may adversely affect the overall ionic balance, with a long chain of cascading effects significantly decreasing newts survival probabilities when environmental pH lowering occurs.
Global biodiversity is declining at an unprecedented rate, and this issue is particularly acute for amphibians that may be the only major group currently at risk at the worldwide scale (Wake and Vredenburg, 2008; Alroy, 2015). Several probable causes have been claimed to explain amphibian populations decline (e.g., habitat loss, increased disease susceptibility, chytridiomycosis caused by amphibian pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, climate changes and its consequences, impacts of exotic species, etc.). Even though multiple factors may have contributed to this phenomenon, it is commonly accepted that the disturbance and degradation of natural environments is playing a major role in determining such negative global outcome (Bruhl et al., 2013; Cerasoli et al., 2017).
From the end of the 70ies, acidification was recognized as a major threat to the environment in Europe and North America (Likens et al., 1979) leading to the ratification, in 1979, of the first regional environmental convention aimed to reduce both nitrogen and sulfur emissions, that are the main cause of acidic deposition (UNECE, 2014). As a result of this international effort, the emissions of these substances have been reduced considerably since the mid-1980s, (Schöpp et al., 2003; Helliwell et al., 2014), thus, positively influencing the chemical quality of surface waters. Despite these achievements, the acidification is still considered a possible cause for amphibian decline (Bradford et al., 1992, Bradford et al., 1994; Blaustein et al., 2003), and remains an environmental issue of great concern (Garmo et al., 2014).
It has been widely demonstrated that an excessive acidification in water bodies exerts harmful effects on wildlife either directly or by increasing the adverse effects of other stressors (Ortiz-Santaliestra et al., 2007). Due to their complex life-cycle and their biological characteristics, amphibians are particularly sensitive to acidification (Wells, 2010; Woodley et al., 2014). Moreover, small permanent or ephemeral ponds, in which many amphibian species live and reproduce, are most vulnerable to the alteration in abiotic parameters, including a lowering of the pH (Rowe et al., 1992). Amphibian larval stages are less tolerant to adverse effects of acidification compared to adults (Pierce, 1993; Woodley et al., 2014). This high sensibility to changes in water conditions is partly due to the highly permeable and uncornified skin, crucial for both gas exchange and osmoregulation (Freda, 1986; Hopkins, 2007).
Although research clearly indicates that acidification continues, implying a significant ecological risk for this vertebrate group, a limited number of studies have been conducted on the effects of acidification in amphibians, compared to other aquatic models.
Earlier literature data on low-pH effects, some of which are rather old, have mainly been dedicated to i) embryonic development and tolerance (Beattie et al., 1992; Grant and Licht, 1993; Räsänen et al., 2002; D’Amen et al., 2007; Ortiz-Santaliestra et al., 2007; Shu et al., 2015), ii) larval sensitivity, survival and developmental traits (Gascon and Bider, 1985; Cummins, 1986; Rowe et al., 1992; Green and Peloquin, 2008; Räsänen et al., 2008), and iii) adult survival and behavior (Horne and Dunson, 1994; Kutka, 1994; Ortiz-Santaliestra et al., 2009).
Surprisingly, only few studies documented the response of the amphibian integument to acidic pH (Linnenbach et al., 1987; Böhmer and Rahmann, 1990) and there remain numerous unknowns on the effects of a sub-lethal exposure to acid during aquatic larval stages. Furthermore, only a single report is available on the morphological and ultrastructural modification in the larval epidermis of anurans following acute lethal exposure to acid water (Meyer et al., 2009). To the best of my knowledge, no previous study has ever investigated the effects of a non-lethal value of acid pH onto amphibian larval skin.
However, in a previous study, we demonstrated that acidification causes harmful effects on survival, behaviour, and gill morphology, inducing a remarkable modification in the expression pattern of cytokeratin Type II in larvae of the newt Triturus italicus (the former name of Lissotriton italicus; Brunelli and Tripepi, 2005). For its role as interface between the body and the environment, the skin is directly exposed to water pollutants, thus, can represent a primary target organ also for low-pH toxicity. In this light, the aim of this study is to first assess, in Lissotriton italicus larvae, the effects that can be induced on epidermal histology after exposure to a non-lethal degree of acid pH. In a second step, to analyse the modifications in the expression pattern of specific cytokeratins (keratin-type II clone AE3 and High Molecular Weight keratins), aquaporin 3 (AQP3), as well as in the inducible isoform of nitric oxide synthase (iNOS) imputable to lowering of pH. Indeed, in amphibians, several environmental factors are likely to result in plasticity in growth rate and development. For this reason, we decided to focus our analysis onto the expression pattern of specific proteins that may reflect morphological and physiological maturation of amphibian epidermis (e.g., proteins involved in mechanical protection, osmoregulation, and respiration mechanisms).
Cytokeratins are members of the intermediate filament family, which are often used as a marker for differentiation and metamorphosis in amphibian skin. In fact, their expression is linked to regulated and specific transformations of both the structure and the transport properties of epithelial tissues that are taking place during ontogenesis (Shimizu-Nishikawa and Miller, 1992; Suzuki et al., 2002; Ishida et al., 2003; Brunelli et al., 2015).
AQP3 is an isoform belonging to the so-called aquaglyceroporin, a sub family of water conducting transmembrane proteins (AQPs). AQP3 is expressed in most types of epithelial cells including the epidermis, contributing to water permeability of biological membranes (Rojek et al., 2008; Boury-Jamot et al., 2009; Verkman, 2011). While some of the AQPs are constitutive, a differential expression is possible for different isoforms, depending on both pathological and physiological events (Brunelli et al., 2007; Chikuma and Verkman, 2008; Hoogewijs et al., 2016; Zhu et al., 2016; Thiagarajah, 2017). Finally, the present study has also evaluated, as inflammatory markers, the expression of the inducible isoform of nitric oxide synthase (iNOS) an early response protein. This protein is usually activated upon injury, promoting the synthesis of NO, a highly versatile and ubiquitous signaling molecule (Xu et al., 2001; Ramyaa et al., 2013; Tomankova et al., 2017).
To the best of my knowledge, the study presented herein is the first report focusing on the morphofunctional alterations induced by a non-lethal acid pH on amphibian larval skin. Furthermore, since few reports throughout the literature have been dedicated to study the effects of acidification in both larvae and adult amphibians, the results presented herein will contribute to the discussion regarding the role of acidification in affecting amphibian populations.
2. Materials and methods
2.1. Study species
The newt Lissotriton italicus is an endemic species distributed throughout most of central and southern Italy. This species, listed in Appendix II of the Bern Convention and included in Annex IV of the EU Habitat Directive, is recorded as Least Concern (Arntzen et al., 2009) in view of its relatively wide distribution and the tolerance of a broad range of habitats, such as woodland and agricultural habitats.
2.2. Collection and animal husbandry
The larvae used in the present study were collected, using a close-mesh net, from a field near the city of Cosenza (Calabria, Southern Italy) during the breeding season, that in Calabria occurs from November to May (Sperone and Tripepi, 2005). The animals were transported to the laboratory, reared in 50 l aquaria filled with water taken from the original pond (pH of 7.2–7.5), and kept at room temperature under a natural light/dark cycle, for a 5-day acclimatization period. The development stage was judged by the presence of certain distinctive morphological features, according to the developmental tables of Gallien and Bidaud (1959).
2.3. Experimental setup and exposure conditions
Dose selections, range finding test, water parameters measurements and chemical analysis were all based on our previous report (Brunelli and Tripepi, 2005). Briefly, the lethal level of pH was determined by exposing the larvae to decreasing pH values. We evaluated that the pH value at which 50% of mortality occurs in 96 hours (LC50) is pH 4.0 for Lissotriton italicuslarvae. During the experiments (all replicates) no mortality occurred neither in the control group nor in the sub-lethal group.
Experiments were initiated when all larvae reached the developmental stage 53–55 a of the Gallien and Bidaud (1959) tables. During the stage 53–55 a, the forelimb and the hind limb have 4 and 5 digits, respectively. The gills are elongated and highly branched.
Groups of five larvae with a mean mass of 1.57 ± 0.2 g, were randomly chosen and moved to 15 l glass tanks (40 cm × 25 cm × 20 cm) containing either natural pond water or pond water previously acidified by addition of HCl drops to reach a final pH of 4.5. The pH level was measured with a Delta OHM HD8705 pH meter with automatic temperature compensation, and measurements were taken every 4 h during day-time and every 8 h during night-time. The pH value (4.5) was regulated by adding water or HCl as required, thereby compensating any pH buffering due to the water ion composition.
For the entire duration of the experiment, animals were held under a natural light/dark photoperiod at room temperature and fed on alternate days with Artemia salina (after thorough washing in fresh water). Three replicate experiments were conducted.
All experimental procedures for animal handling and tissue removal were carried out according to the recommendations of Animal Care and Use Committee and were approved by the Italian State Office of Environment, Rome, Italy. Permit Number: 2004/30911. All remained animals not used for the experiments were released at the place of collection.
2.4. Morpho-functional analysis of the skin
The skin was removed from both exposed and control larvae after 24, 48, and 192 h respectively. At the time of sacrifice, the animals were deeply anesthetized with tricaine methane sulfonate (MS-222, 0.1% – Sigma-Aldrich Chemicals Co., St. Louis, MO). Small portions of skin (2–5 mm), including the underlying dermal and muscular layers, were quickly excised from three different dorsal regions: behind the head, in the median region, and at the base of the caudal fin. After the removal and dissection phases, samples from each animal were used for both conventional light microscopy (LM) and immunohistochemical analysis.
2.4.1. Light microscopy (LM)
After skin removal, samples were placed in 4% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS 0.1 M, pH 7.2, 4 °C) for 48 hours and subjected to a post-fixation in osmium tetroxide (1% in PBS). At the end of the fixation phase, samples were dehydrated in an increasing series of ethanol, soaked in propylene oxide, embedded in Epon-Araldite (Araldite 502/Embed 812, Electron MicroscopySciences) and cut using a Leica UltraCut UCT (Leica Microsystems, Wetzlar, Germany). Semi-thin sections (1–2 μm) for light microscopy were stained according to the trichrome method of Humphrey and Pittman (1974) and were examined with a LM Leitz Dialux 20 EB (Leica Microsystems).
For immunohistochemistry analysis and after a fixation in Bouin liquid for 24 hours, skin samples were dehydrated in an increasing series of ethanol solutions, cleared in xylene, and finally embedded in paraffin wax. Sections of 10 μm were deparaffinized and subjected to the indirect immunofluorescence technique (Coons et al., 1995). Samples were washed in a phosphate buffer (PBS) and then incubated for 10 min in a moist chamber with 20% of the species-appropriate normal serum (Sigma-Aldrich Chemical Co.) to block non-specific sites. Unwashed sections were incubated overnight at 4 °C with following primary antisera: anti-cytokeratin HMW developed in mouse (that recognized cytokeratin 66, 57, 51, 49 kDa corresponding to K1, K5, K10, K11; Gown and Vogel, 1982), anti-keratintype II clone AE3 developed in mouse, anti-nitric oxide synthase inducible developed in mouse, anti- water channel aquaporin 3 developed in rabbit (concentrations and suppliers as indicated in Table 1). The next day, slides were washed in PBS and the primary antibodies were visualized through an incubation of 30 min at room temperature in the dark with corresponding fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and FITC-conjugated sheep anti-rabbit IgG (Sigma-Aldrich Chemical Co., St. Louis, MO, USA), at working dilutions of 1:100. After several washes in PBS, sections were counterstained with propidium iodide for 30 s (Sigma-Aldrich Chemical Co.; 1:200 in PBS) which stains mainly DNA, and to a lesser extent RNA. After a final wash step in PBS, slides were finally mounted. Immunolabeling specificity was verified by substituting the primary antibody with non-immune normal serum. Specimens were examined with a Leica TCS SP2 Confocal Laser Scanning Microscope (Leica Microsystems).
|Mouse anti-cytokeratin HMW||Monoclonal||Dako Cytomation, Carpinteria, CA, USA||1:100|
|Mouse anti-keratintype II clone AE3||Monoclonal||Progen, Heidelberg, Germany||1:100|
|Mouse anti-nitric oxide synthase, inducible, clone NOS-IN||Monoclonal||Sigma–Aldrich Chemical Co||1:100|
|Rabbit anti- water channel aquaporin 3||Polyclonal||Sigma–Aldrich Chemical Co||1:100|
3.1.1. Control group
The histological organization of L. italicus larval skin, under normal conditions, has been previously described (Brunelli et al., 2007, Brunelli et al., 2015; Perrotta et al. 2012), and only a brief general description relevant to the present paper will be given. The general epidermis arrangement of untreated larvae was similar to that of other Urodela species with an organization in distinct layers above the supportive connective tissue. The more external layer consisted of flattened pavement cells (PVCs) and few scattered mitochondria-rich cells (MRCs), pear-shaped, and with a narrow apical surface. Basal cells (BCs) formed a continuous layer connected to the basal lamina, large Leydig cells (LCs), rounded or ovoid in shape, formed up to 3 intermediate layers. These LCs were easily recognizable by their clear cytoplasm filled with secretory granules and vacuoles (Fig. 1A).
3.1.2. pH-exposed group
After 24 h of exposure to low pH (4.5) the general arrangement of the epidermis was still maintained and it was possible to recognize the well-distinct layers. However, LM observations revealed that in the outer layer, PVCs showed an extensive loss of cytoplasmic detail (pale-staining and poor cytoplasm typical of necrotic cells). Under a stereoscopic microscope, the epidermis appeared covered with a dense mucous layer (data not shown) that was partially lost during the preparation of the samples. However, also under LM the active secretion of mucus was evident outside the apical surface (Fig. 1B). Histological modifications became more evident after 48 h of exposure. In all treated samples, the whole epithelium appeared thinned and flattened compared with that of the control specimens. The external layer was strongly stained and both the cytoplasmic and nuclear contents of PVCs became indistinguishable. Noteworthy, in more than one area, it was possible to observe the detachment of these cells from the epithelial layers below. A darkened replacement layer could be seen underneath the detaching external layer. Leydig cells were fewer in number, and wide spaces and lacunae appeared between the clear secretory granules, that under normal conditions were always highly compacted. The secretion of mucus was still noticeable (Fig. 1C). Finally, after 192 h of exposure, the epithelium appeared to be strongly flattened and the thickness of the epithelium was occupied by elongated LCs, in a maximum of two layers. In some areas of the epidermis samples, the LCs showed typical cytoplasmic content whereas in large areas the cytoplasm appeared poor. Wide spaces and lacunae between the granules were observed and often the nucleus occupied the entire cytoplasm while the granules were confined to the periphery of the cell (Fig. 1D). Also a great amount of mucous were detected at epidermal surface (Fig. 1D). Moreover, morphological alterations also involved the dermis and a thickening of basement membrane was observed along with the presence of necrotic cells.
In the epidermis of animals from the control group, AE3 (cytokeratin-type II) was found to be expressed in BCs and the immunoreactivity could be mainly detected in the proximal and lateral portions of the membranes with a moderate labeling intensity (Fig. 2A). After 24 h of exposure to low pH, the intensity of staining slightly increased but the localization remained limited to the basolateral membrane of BCs (Fig. 2B). The intensity of the signal was still moderate after 48 h but the distribution pattern substantially changed and the staining was detected from the inner to the outer strata, reaching also the more external layer (Fig. 2C–D). An enhanced AE3 immunostaining could be observed after 192 h; all the epidermal layers were stained and the labeling was higher in BCs (Fig. 2E–F), (see also Table 2).
The localization of high molecular weight proteins (HMW) in the control larval skin revealed a moderate signal at the basolateral cell membrane of BCs, while supra-basal and outer layers were not or only weakly-stained (Fig. 3A). The immunopositive signal slightly increased after 24 and 48 h of exposure to low pH extending to the whole epithelium (Fig. 3B–C), and reached a maximum value after 192 h of exposure becoming particularly intense both in the basal and in the external layer (Fig. 3D–E) (see also Table 2).
Confocal microscopy observations revealed that AQP3 immunopositive signals was not detectable in sections of skin from the control group (Fig. 4A). On the contrary, after exposure to acid pH, the localization of AQP3 was evident at the basolateral membrane of the cells in all the epidermal layers (Fig. 4B–D). The signal increased with exposure time reaching high intensity after 48 h of exposure (Fig. 4C), (see also Table 2).
Immunolocalization of iNOS was absent from larval epidermis of the control group (Fig. 5A). After exposure to low pH, an intense immunostaining for iNOS could be observed in samples from all exposed groups (Fig. 5B–D). The labeling was always found in basal cells, at membrane level and in their peripheral cytoplasm, and after 192 h of exposure, the iNOS positivity extended to the supra basal layers (Fig. 5D), (see also Table 2).
Ionic composition and pH represent the most important physical factors limiting the distribution of organisms in freshwaters. Both factors are also carefully regulated in the body fluids of aquatic organisms (McCullough and Horwitz, 2010; Serrano et al., 2016). As a consequence, the potential risk of acidification to the aquatic biota has been often associated with a perturbation of the general ionic balance, given its critical importance in homeostasis maintenance. In amphibian larvae, the disturbance of ion regulation may represent an important mechanism through which low pH may exerts its toxicity, whereby sodium loss increases (Freda, 1986; Freda and Dunson, 1984; Meyer et al., 2009; Wells, 2010).
The amphibians skin is a very dynamic organ being the first site of contact with the environment, and, thus, it is easily affected by low-pH toxicity. During the larval phase the skin exerts a prominent role in both respiration and osmoregulation, consequently, morphological and/or functional alterations of this organ may adversely affect the ionic balance, with a long chain of cascading effects (Freda, 1986; Pierce, 1993; Wells, 2010).
To the best of my knowledge, the present study demonstrated for the first time that a short-term exposure to a non-lethal levels of acid pH (4.5) induces severe morphological and functional alterations in amphibian larval epidermis. In L. italicus larvae, the early responses to the low-pH injury were the presence of a conspicuous amount of mucus that covers the outer surface, and the emptying of the LCs cytoplasm. A similar mucus increase has been described in gills of both fish and larval urodeles after exposure to acid stress (Linnenbach et al., 1987; Böhmer and Rahmann, 1990; Brunelli and Tripepi, 2005), whereas Meyer et al. (2009) reported, in tadpoles of L. fallax, an unmodified mucus production after acute lethal acid exposure. The secretion released from the vacuoles of the pavement cells under normal conditions, gives rise to a mucous coating that facilitate the exchange of gases (Kato and Kurihara, 1988). However, in larval urodeles, PVCs are not the only secretory cells and the most prominent feature of the epidermis is the presence of Leydig cells, characterized by numerous secretory granules. Although their role has often been questioned, several lines of evidence have been reported supporting the mucus-secreting function of LCs (Brunelli et al., 2009 and reference therein). In the present experiment, mucus secretion and the concomitant cytoplasm depletion suggest an increased LCs secretion activity that may have led to the reduction of granules and the formation of the external mucus coating.
In this study, histological observations also revealed that the degree of pathological modifications became more pronounced with increased exposure time, and after 48 h of exposure, a pronounced flattening of the epithelium along with the loss of cytoplasmic detail in PVCs was observed. These degenerating PVCs generated a keratinized shedding layer, similar to that observed at metamorphic climax which was then confirmed by immunolocalization of the cytokeratins (Fox, 1986).
During metamorphosis the epidermis of amphibian undergoes marked morphological and physiological changes that include the modification in the expression pattern of a wide array of proteins (Brown and Cai, 2007). When the thin epidermis of larvae is converted in the multilayered epidermis of adults, the expression of cytokeratins changes to acquire a better protection function (Alibardi, 2002, Alibardi, 2006; Brunelli et al., 2015). The present study clearly demonstrates that low pH modifies the cytokeratins expression: after 48 h both AE3 and high MW keratins were highly expressed in the external shedding layer, unlike that observed in untreated samples (Alibardi, 2002, Alibardi, 2006; Brunelli et al., 2015). The strong expression of HMW keratins, a marker of keratinization (Brunelli et al., 2015), suggests an anticipated epidermal maturation. Interestingly, after exposure to low pH, AE3 cytokeratins were not restricted to the innermost epidermal layer but reached the outer layer thus contributing to keratinization. This observation also corroborates our previous results in L. italicus gills (Brunelli and Tripepi, 2005).
The precocious keratinization of the external layer may implicate the need for water supply from the subepithelial compartment, as is the case in adult skin. The active transport of both water and glycerol through the skin is largely controlled by aquaporins (Ishibashi et al., 2011). AQP3 is an isoform localized in many epithelial cells. In the skin it is expressed in the basal keratinocytes acting as a water/glycerol transporter from the subepithelial side to the epithelium above (Matsuzaki et al., 1999; Boury-Jamot et al., 2009; Zhu et al., 2016). In amphibians, AQP3 expression initiates late in development and becomes more intense after metamorphosis, especially during the terrestrial phase when the threat of evaporative water loss becomes severe (Brunelli et al., 2007). The overexpression of AQP3 observed herein as a consequence of acid exposure may represent an attempt of larval skin to counteract the precocious phenomenon of keratinization, prematurely assuming an adult-pattern.
Furthermore, it has been demonstrated in mouse keratinocytes, both in vivo and in vitro, that AQP3 is involved in epidermal cell proliferation (Hara-Chikuma and Verkman, 2008). It has also been shown recently that AQP3-mediated transport of glycerol plays a crucial role in several skin diseases, including tumor growth, wound healing, tumorigenesis, atopic dermatitis and psoriasis (Verkman, 2008; Verkman et al., 2008; Ishibashi et al., 2011; Hara-Chikuma et al., 2015; Zhu et al., 2016; Thiagarajah et al., 2017). In this light it is legitimate to speculate that the increase in AQP3 expression induced by low pH exposure may, in turn, stimulate cell migration and proliferation in amphibian skin.
The inducible isoform of nitric oxide synthase plays a vital role in the skin, orchestrating normal regulatory processes (Lawrence and Brain, 1992; Coffman, 1994; Bishop and Brandhorst, 2001, Bishop and Brandhorst, 2003; Cals-Grierson and Ormerod, 2004). Moreover, it exerts the role of a metamorphosis regulator in amphibian larval epidermis (Brunelli et al., 2005). This study provides evidence that the expression of this enzyme is induced by exposure to low pH in amphibian larval epidermis. This finding supports our hypothesis of an anticipated maturation of skin in treated animals, in agreement with the well-known phenotypic plasticity of amphibians (Boorse and Denver, 2004).
In conclusion the results in the present study suggest that acid exposure activates a complex network of responses from 24 to 192 h of exposure to acidic condition such as mucus secretion, inflammation, cell proliferation and anticipated keratinization. However, a better understanding of the details of the sequence of events in the amphibian response to water acidification still needs more research and finer resolution.
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