Osmoionic homeostasis in bivalve mollusks from different osmotic niches: Physiological patterns and evolutionary perspectives

Comparative Biochemistry and Physiology, Part A 240 (2020) 110582

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Osmoionic homeostasis in bivalve mollusks from different osmotic niches: Physiological patterns and evolutionary perspectives Isadora Porto Martins Medeirosa, Samuel Coelho Fariac,d, Marta Marques Souzaa,b,

T

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a

Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio Grande, FURG, Brazil Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Brazil c Instituto de Biociências, Universidade de São Paulo, USP, Brazil d Department of Evolution, Ecology and Organismal Biology. University of California, Riverside, CA 92521, USA b

ARTICLE INFO

ABSTRACT

Keywords: Invertebrate Osmoconformation Osmolytes Salinity stress Evolution

Physiological knowledge gained from questions focused on the challenges faced and strategies recruited by organisms in their habitats assumes fundamental importance about understanding the ability to survive when subjected to unfavorable situations. In the aquatic environment, salinity is particularly recognized as one of the main abiotic factors that affects the physiology of organisms. Although the physiological patterns and challenges imposed by each occupied environment are distinct, they tend to converge to osmotic oscillations. From a comparative perspective, we aimed to characterize the osmoregulatory patterns of the bivalve mollusks Corbicula largillierti (purple Asian cockle), Erodona mactroides (lagoon cockle), and Amarilladesma mactroides (white clam) inhabitants of different osmotic niches - when submitted to hypo- and/or hyperosmotic salinity variations. We determined the hemolymph osmotic and ionic concentrations, tissue hydration, and the intracellular isosmotic regulation (IIR) from the use of osmolytes (organic and inorganic) after exposure to species-specific salinity intervals. Additionally, we incorporated phylogenetic perspectives to infer and even broaden the understanding about the patterns that comprise the osmoionic physiology of Bivalvia representatives. According to the variables analyzed in the hemolymph, the three species presented a pattern of osmoconformation. Furthermore, both ionic regulation and conformation patterns were observed in freshwater, estuarine, and marine species. The patterns verified experimentally show greater use of inorganic osmolytes compared to the participation of organic molecules, which varied according to the osmotic niche occupied in the IIR for the mantle, adductor muscle, and gills. This finding widens the classic vision about the preferential use of certain osmolytes by animals from distinct niches. Our phylogenetic perspective also indicates that environmental salinity drives physiological trait variations, including hemolymph osmolality and the ion composition of the extracellular fluid (sodium, chloride, magnesium, and calcium). We also highlight the important role played by the shared ancestry, which influences the interspecific variability of the hemolymph K+ in selected representatives of Bivalvia.

1. Introduction Bivalve mollusks are subject to several abiotic variables that may exert significant effects on their biology. Among them, salinity is recognized as an important environmental factor since its fluctuations tend to occur according to the season, tide, and climate (Kinne, 1966; Lynch and Wood, 1966; Berger and Kharazova, 1997). Additionally, anthropogenic intensification and events associated with climate change contribute to increasing the frequency and extent of the natural variabilities (Rivera-Ingraham and Lignot, 2017). Subsisting throughout the aquatic environment, bivalve mollusks

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are found from fresh to sea waters (Matsushima et al., 1987). The species exhibit a limited capacity for osmotic regulation when in dilute environment to a narrow range of osmotic conformation when in concentrated media (Deaton, 2009; Larsen et al., 2014). Although the patterns manifested and the challenges posed by the variations for each environment are distinct, in general they tend to converge to one common feature: osmotic variation. The physiological effects of unfavorable or fluctuating osmotic conditions are extensively reported and reviewed by the scientific community in Bivalvia from different osmotic niches (Beadle, 1957; Berger et al., 1978; Carvalho et al., 2015; Costa and Pritchard, 1978;

Corresponding author at: Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio Grande, FURG, Brazil. E-mail address: [email protected] (M.M. Souza).

https://doi.org/10.1016/j.cbpa.2019.110582 Received 18 April 2019; Received in revised form 5 September 2019; Accepted 17 September 2019 Available online 08 November 2019 1095-6433/ © 2019 Elsevier Inc. All rights reserved.

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Coughlan et al., 2009; Gharbi et al., 2016; Jordan and Deaton, 1999; Larsen et al., 2014; Pierce, 1970; Ruiz and Souza, 2008; Shumway, 1977). In this context, perturbations caused to hemolymph osmolality, tissue water content, and cellular volume (De Lisle and Roberts, 1988; Larsen et al., 2014; McFarland et al., 2013) might affect homeostasis maintenance. Therefore, considering homeostasis as a state of fundamental stability, one can infer that a wide diversity of physiological mechanisms involved in water and salt balance evolved in the metazoans (Deaton, 2009; Larsen et al., 2014; Zinchenko and Golovatyuk, 2013), especially in groups that occupy variable environments. Among these strategies, intracellular isosmotic regulation (IIR) is a primary physiological adaptive tactic to cope with osmotic fluctuations in the external environment (Matsushima et al., 1987). It specifically activates mechanisms involved in cell volume maintenance. From the adjustment of osmotically active solutes (i.e., ions and amino acids), regulatory events are initiated according to environmental salinity (Deaton and Pierce Jr, 1994; Koivusalo et al., 2009; Pierce, 1982; Wijayasinghe et al., 2017). The IIR comprises fundamental mechanisms associated with regulatory volume decrease (RVD) in hypoosmotic scenarios and strategies that encompass regulatory volume increase (RVI) in cellular shrinkage situations. Due to the importance of the pool of osmotically active solutes during cell volume regulation, as well as their particularities according to the experienced saline stress, the scientific literature includes myriad investigations from this perspective (see Allen and Garrett, 1972; Babarro et al., 2011; Baginski and Pierce Jr, 1978; Burg et al., 2007; Hosoi et al., 2003; Ivanovici et al., 1981; Kube et al., 2006; Matsushima et al., 1987; Pierce Jr, 1971; Potts, 1968). However, it is important to emphasize that such studies tend to focus only on the effects of a specific osmotic shock (hypo- or hyperosmotic), and they are usually based on a one-time evaluation of a single class of effectors (inorganic or organic). Further, the literature tends to assume the preferential use of osmolytes according to the environment, as in the case of free amino acids (FAA) in the marine habitat (Yancey, 2005). Moreover, distant from a comparative context within and with specific interests, traditional thinking suggests that a graduated series of successively stronger adaptive mechanisms may have driven the occupancy of increasingly diluted osmotic niches. However, correlating these patterns exclusively to the environmental effect is a misguided practice in the study of comparative physiology (Garland et al., 2005; McNamara and Faria, 2012; Faria et al., 2017a, 2017b). An alternative to this conception is the incorporation of an extended time scale, specifically the inclusion of the systematics for the investigated groups. This addition allows evaluation of physiological transformations and their underlying evolutionary processes. Since the phylophysiological approach also presupposes that species are temporally linked, a factor that can confer phylogenetic structuring (Garland et al., 2005), it should be considered in studies with comparative bias. In this light, we aimed to identify the main osmotic effectors recruited by the IIR (when subjected to salinity fluctuations) in three bivalve mollusks: the freshwater Corbicula largillierti (purple Asian cockle), estuarine Erodona mactroides (lagoon cockle), and marine Amarilladesma mactroides (white clam). All of these species inhabit different osmotic niches. Thus, (I) we characterized experimentally intracellular isosmotic regulation by means of organic and inorganic solutes, on exposure to hyper- and hypoosmotic media, and (II) partially retrieve the evolutionary history of osmoregulation by detecting interspecific physiological patterns in the Bivalvia class.

and the marine A. mactroides Deshayes, 1854 (white clam; 32°02′28.89"S, 52°12′97.98"W) in the State of Rio Grande do Sul, Brazil. The animals were then transported to the Instituto de Ciências Biológicas of the Universidade Federal do Rio Grande – FURG (Rio Grande, RS, Brazil) and acclimated for 7 days in 20-L plastic tanks. They were subjected to constant aeration, ambient temperature (≈ 21 °C), a 12-h light:12-h dark photoperiod, and feeding three times a week with microalgae at their respective natural habitat salinities (0‰ for C. largillierti, 11‰ for E. mactroides [according to the daily monitoring of the Port of Rio Grande - www.portosrs.com.br/site/responsabilidade_ ambiental_consultas_publicas.php], and 28‰ for A. mactroides; Odebrecht et al., 2010). All saline solutions were obtained from the addition of sea salt (Marineland®) to fresh water, or by mixing sea water with fresh water. Salinities were verified using a manual optical refractometer (model K52–100, KASVI). 2.2. Physiological experiments 2.2.1. Osmotic shock exposure The osmotic shock experiment was performed following previously conducted survival trials. The aim was to establish safe salinity ranges for each evaluated species. Thus, the bivalve mollusks were subjected to 96-h osmotic shock that was 25% above and/or below the acclimation salinity (C. largillierti, N = 5; 0, 5, 10, and 15‰; E. mactroides, N = 7; 8, 11, and 14‰; A. mactroides, N = 7; 21, 28, and 35‰). Under the same laboratory conditions and constant aeration, the organisms were directly exposed to such osmotic media in 500-mL plastic containers. During the treatment exposure interval, the animals were not fed. Subsequently, biological material was sampled from the specimens. Hemolymph was drawn from the adductor muscle, the animals were killed by cryoanesthesia, and the gills, mantle, and the adductor muscle were removed and carefully weighed (AG2204, Gehaka; XP6, Mettler Toledo). All samples were immediately stored at −20 °C. 2.2.2. Osmolality and tissue hydration To characterize the osmoregulatory capacity of each species, hemolymph osmolality (mOsm/kg H2O) was measured using a vapor pressure osmometer (Vapro 5600, Wescor). Additionally, osmolality measures were performed at the treatment salinities. The water content of the mantle, adductor muscle, and gills from each species was measured by weighing fresh tissue samples on an electronic balance (AG2204, Gehaka; XP6, Mettler Toledo) and then reweighing it after oven drying at 60 °C for 72-h. From the obtained data, the tissue hydration (TH, %) was calculated using the following formula: (wet mass dry mass ) TH = × 100 . wet mass 2.2.3. Osmolyte concentration The concentration of inorganic osmolytes such as Na+ and K+ from the experimental media, hemolymph, and tissues was detected by flame photometry, as adapted from Amado et al. (2006). The [Cl−] was measured using a commercial kit (Colorimetric Chlorides, Doles Reagents). Each ion concentration was calculated using the standard curve equation. Data are expressed as mM or mM/mg dry mass and later relativized for percentage (%). For organic osmolytes, FAA analysis was performed using a fluorescence spectrophotometer (FilterMax F5 model, Molecular Devices), following Fisher et al. (2001) with modifications. The required standard mixtures were obtained using the amino acids taurine, alanine, and glycine. As noted by myriad authors, these three organic osmolytes are usually involved in maintaining the osmotic equilibrium of bivalves (distributed from fresh to sea water), since large amounts of these compounds are found in the FAA pool in different tissues (Babarro et al., 2011; Burg and Ferraris, 2008; Deaton et al., 1989; Fyhn, 1976; Gilles, 1978; Henry et al., 1980; Hosoi et al., 2008; Jordan and Deaton, 1999; Livingstone et al., 1979; Otto and Pierce, 1981; Pierce and

2. Materialandmethods 2.1. Collection and acclimation of animals We sampled specimens of the freshwater C. largillierti Philippi, 1844 (purple Asian cockle; 32°16′21.07"S, 53°99′32.36"W), the estuarine E. mactroides Bosc, 1802 (lagoon cockle; 32°02′28.89"S, 52°12′97.98"W), 2

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Amende, 1981). Taurine, alanine, and glycine ratios in bivalves, respectively, were established from our literature review (see section 2.3.2) as follows: freshwater (60%, 20%, and 20%), estuarine (20%, 60%, and 20%), and marine (70%, 20%, and 10%). Thus, standard mixtures were prepared at 40, 4, 0.4, 0.04, and 0.004 μM to integrate the standard curve used in the test. The FAA concentration calculation was performed using the equation of the line reached by reading a standard curve established for each osmotic niche. The FAA concentration was obtained in μM/mg wet mass, and the conversion to μM /mg dry mass was done using the following formula adapted from Faria et al. (2011):

Dry mass =

(100

mactroides are not present in the phylogenies assumed here, the environment occupied by them is sampled in the meta-analysis. After applying this selection method, and matching species with phylogeny for each physiological trait, the physiological data from 18 publications were compiled to compose the meta-analysis (Table 1). The phylogenetic signal, i.e., the tendency of closely related species to resemble each other more than randomly drawn species (Münkemüller et al., 2012), was measured by employing an autocorrelation analysis using Moran's I index (Diniz-Filho, 2001; Gittleman and Kot, 1990), which varies between −1 and + 1. Significant positive values describe high similarity between closely related species, while significant negative values mean that related species are dissimilar. We present Moran's I only for hemolymph osmolality and ion concentration because of the higher number of taxa with available data. Phylogenetic generalized least squares (PGLS) was applied to test the evolutionary associations between salinity and some osmoregulatory traits. PGLS predicts that the residual variation among species is correlated, as expected due to the shared inheritance (Garland and Ives, 2000; Grafen, 1989; Lavin et al., 2008). The α parameter of the Ornstein-Uhlenbeck (OeU) evolutionary model (Butler and King, 2004), an indicator of the selection strength that forces a trait value to its optimum, was simultaneously calculated in order to fit the best evolutionary model. α = 0 means that covariance among species is a consequence of random evolutionary changes, as modeled by the Brownian motion model (Butler and King, 2004). The evolutionary associations were presented by applying a Maximum Likelihood Method (Felsenstein, 1985), which describes the ancestral states of each node and also illustrates the extant species values. The analyses were performed using the R (R Core Team 2015) packages nlme (Pinheiro et al., 2016), picante (Kembel et al., 2010), geiger (Harmon et al., 2008), and phytools (Revell, 2012); the minimum significance level was set at p = .05.

wet mass tissue hydration)

Data are expressed as μM /mg dry mass, later relativized for percentage (%). 2.3. Statistical analyses 2.3.1. Intraspecific analysis Since some variables were not normally distributed, the data were submitted to non-parametric analysis using the Kruskal-Wallis H test, followed by Dunn's post-hoc test to detect statistically different means (p ≤ .05) or the Mann-Whitney Utest for independent samples (physiological versus osmotic and ionic concentrations of the exposure solutions). Data are expressed as mean + standard error of the mean (SEM). 2.3.2. Interspecific analysis Some data related to bivalve osmoregulation (hemolymph osmolality, extracellular [Na+], [Cl−], and [K+], and FAA tissue concentrations) were compiled through a systematic review (Leenaars et al., 2012), in a meta-analytic approach, using three databases (PubMed, Scopus, and Web of Science). The chosen findings ensure the relationship of homology between experiments and species. A total of 933 papers were initially obtained from different databases (PubMed 22 returns, Scopus - 849 returns, and Web of Science - 62 returns). The molecular phylogenetic hypotheses assumed in this study were obtained from Time Tree computational tool (www.timetree.org), a searchable public knowledge base that gathers information about the evolutionary time scale of life, based on 3164 studies and that include 97,085 species (Kumar et al., 2017). Problems regarding species names were carefully solved using the Worldwide Mollusc Species Data Base (WMSDB) database. Although C. largillierti, E. mactroides, and A.

3. Results 3.1. Osmolality measurement Hemolymph osmolality of C. largillierti at 15‰ salinity was higher compared to the acclimation condition (0‰; H = 12.20; p < .05; Fig. 1a). When compared to the external osmolality, the purple Asian cockle hemolymph osmotic concentration at acclimation was higher (50.33 ± 18.35 mOsm/kg H₂O; U = 1; p < .05). On the other hand, E. mactroides and A. mactroides hemolymph osmolalities were different between hypo- and hyperosmotic conditions (3.44 ≤ H ≤ 17.82;

Table 1 Osmoregulatory traits compiled from the literature and used in the comparative analyses. Speciesa

Niche

Osmolality hemolymph (mOsm/kg H₂O)

[Na+] mM

[K+] mM

[Cl−] mM

[Mg2+] mM

[Ca2+] mM

Reference

Corbicula fluminea Dreissena polymorpha

FW FW

63.2 42.66

31.47 17.33

0.952 0.5

25.99 17.13

– 0.9

– 4.06

Pinctada margaritifera Sinanodonta woodiana MytiIlopsis leucophaeata Aequipecten opercularis Callista chione Crassostrea gigas Crassostrea virginica Modiolus modiolus Mya arenaria Mytilus edulis

FW FW BW SW SW SW SW SW SW SW

31.8 45 50 970 1109 980 940 980 1015 1003

14.7 15.8 24 470 470 473.05 – 471 593 477

0.37 0.45 1.9 – – 12 – – 12 20

– 13.7 20 – – – – – 520 520.33

0.41 – 3 53 – 54.4 – 54.1 50 58

1.86 – 2.1 10.2 – 10.2 – 10.2 10.6 10.4

Perna perna Perna viridis

SW SW

990 950

400 –

56 –

420 –

– –

– –

Ruiz and Souza, 2008 Byrne and Dietz, 2006; Dietz et al., 1996; Dietz et al., 1997 Shakhmatova et al., 2006 Matsushima and Kado, 1982 Deaton et al., 1989 Shumway, 1977 Zatta and Cervellin, 1987 Knowles et al., 2014; Shumway, 1977 McFarland et al., 2013 Shumway, 1977 Deaton, 1992; Shumway, 1977 Costa and Pritchard, 1978; Hoyaux et al., 1976; Lange, 1963; Livingstone et al., 1979; Shumway, 1977 Rola et al., 2017 McFarland et al., 2013

a Scientific name: some species have undergone nomenclature updates. Thus, the names highlighted in bold have been corrected according to the current nomenclature.

3

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(0 ≥ U ≤ 10; p < .05). The [Na+] and [Cl−] in E. mactroides and A. mactroides were different between hypo- and hyperosmotic conditions (10.54 ≤ H ≤ 16.04; p < .05; Fig. 2d,f,g,i), but did not differ when compared to the acclimation salinity (Fig. 2). The [K+] for both species did not vary with 96-h exposure to the differ salinities (3.35 ≤ H ≤ 4.99; p > .05; Fig. 2e-h). When comparing the ionic environmental concentrations, the hemolymph [Cl−] was lower at acclimation (11‰, brackish water) and hyperosmotic conditions (14‰, brackish water and 35‰, sea water; 0 ≥ U ≤ 3; p < .05). On the other hand, the [Cl−] increased in E. mactroides fluid after 96-h hypoosmosis. The [K+] in A. mactroides increased under hypoosmotic salinity (21‰, sea water) compared to the environmental salinity (U = 3; p < .05). Finally, the hemolymph [Na+] was higher in the E. mactroides fluid under hypoosmotic stress (8‰), while it was lower at 28‰ salinity after 96-h exposure in A. mactroides (U = 1; p < .05). 3.3. Tissue hydration The water content of the mantle, adductor muscle, and gills of the freshwater bivalve (C. largillierti) varied after 96-h exposure to the hyperosmotic conditions (7.86 ≥ H ≤ 11.44; p < .05; Fig. 3a-c). There was a decrease in hydration relative to the acclimation condition in the mantle at 5‰, the adductor muscle at 10‰, and the gills exposed to 15‰ salinity. In the estuarine species (E. mactroides), only the gill hydration after 96-h exposure was different between hypo- and hyperosmotic conditions, with a decrease in this parameter at 14‰ compared to 8‰ salinity (H = 6.14; p < .05; Fig. 3c). At 35‰ salinity, there was a decrease in the mantle water content for the marine bivalve (A. mactroides) when compared to 21‰ salinity (H = 8.15; p < .05; Fig. 3a). In contrast, E. mactroides and A. mactroides presented no variation in the adductor muscle or gill water content (0.48 ≥ H ≤ 4.50; p > .05; Fig. 3b and c). 3.4. Inorganic osmolytes in tissues The tissue ionic content varied according to the salinities to which the bivalves were exposed. Compared to acclimation, the C. largillierti mantle [Na+] was higher when submitted to 10–15‰ salinity; [Na+] was also elevated in the gills after 15‰ exposure (Fig. 4a). The mantle [K+] and [Cl−] increased at 10‰ salinity compared to acclimation (Fig. 4b and c). The same effect was observed for the adductor muscle and gills when exposed to higher salinity (15‰; 0.22 ≤ H ≤ 15.79; p < .05). In the estuarine E. mactroides, the mantle [Na+] increased in hyperosmotic relative to hypoosmotic salinity (H = 6.41; p < .05), but did not differ when compared to acclimation (Fig. 5a). After 96-h exposure, the mantle also exhibited an increase in [K+] when under hypoosmotic shock compared to acclimation (H = 7.53; p < .05; Fig. 5b). In the marine A. mactroides, the mantle [Na+] was different under hyperosmosis compared to the other salinity treatments (H = 13.62; p < .05; Fig. 6a). In adductor muscle and gills, after 96-h exposure the [Na+] ranged between the osmotic shocks (hypo- and hyperosmosis; 8.21 ≤ H ≤ 9.57; p < .05). The [K+] only increased in the gills during hyperosmotic stress (H = 8.83; p < .05; Fig. 6b). In contrast, the [Cl−] of E. mactroides and A. mactroides tissues did not vary between the investigated salinity treatments (1.12 ≤ H ≤ 4.28; p > .05; Fig. 5c and 6c).

Fig. 1. Changes in the hemolymph osmolality of (a) C. largillierti (N = 3–4), (b) E. mactroides (N = 4–6), and (c) A. mactroides (N = 6–7) after 96-h exposure to different salinity. Values are expressed as the mean ± SEM. Significant differences (p < .05) are indicated by different letters. No significant differences (H = 3.442; p > .05) were observed for the E. mactroides specimens for 96-h exposures. ⋆ represents differences between hemolymph osmolality and external salinity (p < .05).

p < .05) but did differ when compared to the acclimation salinity (Fig. 1b and c). When compared to environmental osmolality, the A. mactroides hemolymph osmotic concentration was lower at 21‰ salinity (U = 0; p < .05). Comparatively, E. mactroides did not show a difference in osmolality of their fluids after 96-h exposure to different salinities and did not differ in the other conducted comparisons (1 ≤ U ≤ 2; p > .05). 3.2. Inorganic osmolytes in the hemolymph

3.5. Organic osmolytes in tissues

The hemolymph ion concentrations for C. largillierti, E. mactroides, and A. mactroides varied according to the exposure salinities (Fig. 2). In the freshwater C. largillierti, the [Na+] increased after 96-h exposure to 10 or 15‰ salinity; [K+] and [Cl−] concentrations increased only in 15‰ (16.64 ≤ H ≤ 17.90; p < .05; Fig. 2a-c). Compared to the ionic environmental concentrations, only [Na+] and [Cl−] were smaller in the hemolymph when the bivalve were exposure to salinities above zero

The FAA concentration in C. largillierti tissues varied according to the osmotic condition to which they were submitted (10.63 ≤ H ≤ 11.92; p < .05; Fig. 7a). Mantle and adductor muscle presented higher FAA amounts in 15‰ salinity compared to acclimation (0‰). In the gills, FAA content decreased in 5‰ salinity exposure compared to 15‰. In E. mactroides, only the gill FAA content was 4

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Fig. 2. Concentration of inorganic osmolytes (Na+, Cl− and K+) in the hemolymph of the (a-c) freshwater C. largillierti (N = 5), (d-f) estuarine E. mactroides (N = 4–5), and (g-i) marine A. mactroides (N = 6–7) after 96-h exposure to osmotic shock. Data are expressed as mean + SEM. Statistical analyses were performed separately for each ion and species. Significant differences (p < .05) are indicated by different letters. No statistically significant differences (p > .05) were observed in [K+] for E. mactroides or A. mactroides hemolymph. Stars (⋆) represent differences between hemolymph and external ion concentrations (p < .05).

significantly different, since both osmotic shock conditions reduced the concentration compared to the acclimation content (H = 5.32; p < .05; Fig. 7b). Comparatively, the marine species A. mactroides only exhibited a significant difference in FAA content in the adductor muscle (H = 11.06; p < .05; Fig. 7c). Specifically, 96-h hyperosmotic exposure increased FAA compared to the other conditions. FAA content in the E. mactroides mantle and adductor muscle (Fig. 7b) and A. mactroides mantle and gills did not vary depending upon the salinity exposure (Fig. 7c; 0.15 ≤ H ≤ 5.45; p > .05).

hemolymph osmolality (31.8 mOsm/kg H₂O) and a lower habitat salinity (0‰) compared to the ancestral hemolymph osmolality estimated at 745 mOsm/kg H₂O, as well the osmolality of the ancestral habitat estimated at 24‰ (706 mOsm/kg H₂O). Similarly, we realized that species with lower [Na+] tend to also show lower [Cl−] (Fig. 9). This phenomenon was observed after conducting an evolutionary correlation analysis between hemolymph [Na+] and [Cl−] (PGLS, F = 314.9; p < .0001; ɑ = 0.83); these ions are the major inorganic hemolymph fluid osmolytes.

3.6. Comparative analyses

4. Discussion

The presence of a phylogenetic signal was significant only for hemolymph [K+] (I = 0.18; p = .02), data that indicate closely related species show similar [K+]. On the other hand, hemolymph osmolality, [Na+], [Ca2+], [Mg2+], and [Cl−] were not significantly related to phylogeny (−0.14 ≤ I ≤ 0.31; p > .05). Concerning habitat salinity, the evaluated species did not manifest similar values (0.06 = I ≤ 0.19; 0.09 = p ≤ .35), a result that suggests higher osmotic niche diversification. Such absence of a significant phylogenetic effect is reinforced by the evolutionary correlation between all physiological traits with habitat salinity (PGLS, 6.5 ≤ F ≤ 862.7; p ≤ .03; 1.9 ≤ α ≤ 93.4): species from higher salinities tend to show larger osmolality values (Fig. 8). It is important to note, however, that some clades show phylogenetic structuring, e.g., the clades formed by Mytilus edulis, Perna perna, Modiolus modiolus, Crassostrea gigas, Crassostrea virginica, Pinctada margaritifera, and Aequipecten opercularis. In this lineage, there is strong consistency among hemolymph osmolality values (940–1003 mOsm/kg H₂O). The exception is P. margaritifera, which shows a lower

From a comparative overview of the osmotic scenarios and the physiological responses expressed by the freshwater C. largillierti, estuarine E. mactroides, and marine A. mactroides, we observed and characterized the different osmoregulatory patterns manifested by these species. With regards to the relationship between external salinity and survival capacity, C. largillierti showed 100% survival against the tested osmotic gradient (0–15‰; data not shown). This finding departs from the reported physiological patterns for many freshwater bivalves, which are extremely sensitive to the hyperosmotic environment, and cannot survive in salinities > 200 mOsm/kg H₂O (≈ 7‰; Dietz et al., 1998; Jordan and Deaton, 1999). However, species of the genus Corbicula constitute an exception to this rule (Dietz et al., 1998), which is in agreement with the observations of Deaton (1981) for Corbicula fluminea, another species with greater tolerance to salinity (400 mOsm/kg H₂O; ≈ 13.6‰). Although the salinity ranges within which organisms can survive differs among species, the Asian cockle (C. fluminea) and purple Asian 5

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Fig. 3. Variations in the water content of (a) mantle, (b) adductor muscle, and (c) gills of C. largillierti (smooth bars; N = 3–5), E. mactroides (dotted bars; N = 4–5), and A. mactroides (striped bars; N = 5–7) after 96-h exposure to osmotic shock. Values are expressed as mean + SEM. Statistical analyses were performed separately for each tissue and species. Significant differences (p < .05) are indicated by different letters. There were no significant differences (p > .05) in the E. mactroides mantle and adductor muscle or A. mactroides gills and adductor muscle after osmotic shock.

Fig. 4. Concentration of inorganic osmolytes (Na+, K+ and Cl−) in the (a) mantle, (b) adductor muscle, and (c) gills of the freshwater C. largillierti (N = 3–5) after 96-h exposure to osmotic shock. Data are expressed as percentage + SEM. Statistical analyses were performed separately for each ion and tissue. Significant differences (p < .05) are indicated by different letters. There was no statistically significant difference (p > .05) in the adductor muscle [Na+].

cockle (C. largillierti) appear to have a set of similar physiological, ecological, and biological characteristics that promote their adaptive success to resist the environmental stress they experience. Additionally, from an evolutionary point of view, our reconstruction of the ancestral state for habitat osmolality inferred 16.7‰ salinity faced by the C. fluminea ancestor (Fig. 8). In the same genus, the observations obtained for C. largillierti regarding survival in hyperosmotic conditions, as proposed by Dietz (1979) for the Corbicula manilensis tolerance capacity to

high salinity, are supported due to an ancestry in brackish waters. Estuarine and marine bivalves (especially inhabitants of coastal regions) are often exposed to salinity changes caused by tidal cycles or rainy periods. Thus, there are significant osmotic gradients between the environment and the organism (Verdelhos et al., 2015). With the ultimate aim of ensuring the survival of species that occupy such variable niches, strategies such as a tolerance limit presented by these animals 6

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Fig. 5. Concentration of inorganic osmolytes (Na , K and Cl ) in the (a) mantle, (b) adductor muscle, and (c) gills of the estuarine E. mactroides (N = 3–5) after 96-h exposure to osmotic shock. Data are expressed as percentage + SEM. Statistical analyses were performed separately for each ion and tissue. Significant differences (p < .05) are indicated by different letters. There were no statistically significant differences (p > .05) in the adductor [Na +] muscle, as well as in adductor and gill [K +]. Additionally, the [Cl−] was not statistically significant in any of the tissues.

Fig. 6. Concentration of inorganic osmolytes (Na+, K+ and Cl−) in the (a) mantle, (b) adductor muscle, and (c) gills of the marine A. mactroides (N = 5–7) after 96-h exposure to osmotic shocks. Data are expressed as percentage + SEM. Statistical analyses were performed separately for each ion and tissue. Significant differences (p < .05) are indicated by different letters. There were no statistically significant differences (p > .05) for the [K+] in the mantle and adductor muscle. Additionally, the [Cl−] was not significantly different in the mantle, adductor muscle, and gills when exposed to osmotic shock.

are fundamental in coping with salinity changes (Nie et al., 2017). In this sense, we observed in E. mactroides and A. mactroides a characteristic euryhaline profile, denoted by > 50% survival after acute exposure to 0–25‰ and 7–56‰ salinity, respectively (data not shown). Given the ability to withstand wide ranges of environmental osmotic concentrations, euryhalinity may be due to lower extracellular fluid variation, greater tissue tolerance to critical salinity, or a lower tissue

permeability to these changes (Kinne, 1966). Turning to the extracellular compartment, specifically the hemolymph osmotic concentration, after 96-h exposure C. largillierti was characterized as an osmotic conformer at salinities above zero. On the other hand, hemolymph osmolality was slightly hyperregulated in fresh water (50.33 ± 18.35 mOsm/kg H₂O; Fig. 1a). This phenomenon is an emblematic pattern for freshwater bivalves (Deaton, 2009; Larsen et al.,

+

+

−

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authors about the limited cellular volume regulatory capacity presented by freshwater bivalve mollusks (Dietz et al., 1997; Gainey Jr. and Greenberg, 1977; Murphy and Dietz, 1976; Potts, 1968). Thus, changes in tissue/cellular volume are understood as indicative of distortion in the steady-state equilibrium of the inflow and outflow of water and salts (Kinne, 1966; Strange, 2004). From the physiological point of view, marine and estuarine bivalves are known osmoconformers (Larsen et al., 2014; Mantel and Farmer, 1983). We verified the classical physiological pattern of osmotic conformation in E. mactroides when submitted to salinity variations (Fig. 1b). Likewise, the A. mactroides hemolymph osmotic concentration was similar to the environmental concentration, except when confronted with hypoosmotic shock, where hemolymph osmolality was slightly below the ambient concentration (hypoconformed) (Fig. 1c). Since changes in water content are considered to be traditional indicators of volume regulation (Amado et al., 2011; Freire et al., 2013; Oglesby, 1981), we also evaluated in E. mactroides and A. mactroides the possible effects of hemolymph osmotic concentration changes on tissue hydration. Our results revealed that, in view of the osmotic shock, the mantle, adductor muscle, and gills from both species maintained the volume they had before the imposed salinity variations (Fig. 3a-c). Although we observed differences in E. mactroides gills and A. mactroides mantle hydration when exposed to larger osmotic intervals (6‰ and 14‰, respectively), such differences appear to be punctual. Additionally, we believe that because they are organs in constant interface with the external environment, they tend to suffer more directly from osmotic changes. To understand the effects of salinity and phylogeny on interspecific variation, we applied comparative phylogenetic methods. This analysis revealed that hemolymph osmolality in natural salinities was not phylogenetically structured, a finding that indicates this characteristic varied randomly among the evaluated species. This observation is reinforced through the evolutionary correlation with habitat osmolality (Fig. 8). Based on this analysis, we infer that salinity boosted the osmotic variability of the hemolymph fluid, since species that occupy environments with higher salinities tend to present, in parallel, greater hemolymph osmolality. It is important to note that despite the absence of a significant effect of the phylogenetic signal for this physiological trait, some clades point to phylogenetic structuring. On the other hand, the P. margaritifera profile (hemolymph osmolality = 31.8 mOsm/kg H₂O; habitat osmolality = 0‰, compared to the estimated ancestral hemolymph osmolality of 745 mOsm/kg H2O and ancestral habitat osmolality of 24‰), possibly indicates that this species occupies more diluted osmotic niches. This finding indicates a deviation of the expected trend for the clade in question (composed mostly of marine species). With regards to hemolymph ionic concentrations, both ionoregulation patterns as ionoconformation were experimentally verified in freshwater (Fig. 2a-c), estuarine (Fig. 2d-f), and marine species (Fig. 2gi). In general, C. largillierti, E. mactroides, and A. mactroides hyporegulated the hemolymph [Cl−] during hyperosmotic saline exposure; C. largillierti concomitantly hyporregulated [Na+] (only at 10 and 15%₀ salinity). Additionally, under hyperosmotic shock, the three bivalves ionoconformed the [K+], just as E. mactroides and A. mactroides also conformed the [Na+], phenomena that conferred similarity between the environmental and extracellular fluid ion concentrations. On the other hand, when confronted with acute hypoosmotic conditions, the estuarine and marine species manifested different regulation and ion conformation patterns. Thus, E. mactroides maintained slightly higher [Na+] and [Cl−], whereas A. mactroides only regulated [K+]. Based on these results, we infer that the maintenance of the evaluated inorganic ions appears to be subject to a specific physiological control, taking into account the osmotic shock experienced. This ability is species-specific or defined according to the occupied aquatic niche. In order to understand the general homeostasis patterns in relation to what is available in the osmoionic literature for bivalves, our

Fig. 7. Concentration of organic osmolytes (taurine, alanine, and glycine) in the mantle, adductor muscle and gills of the (a) freshwater C. largillierti (N = 3–5), (b) estuarine E. mactroides (N = 3–4), and (c) marine A. mactroides (N = 4–6) after 96-h exposure to osmotic shock. Data are expressed as percentage + SEM. Statistical analyses were performed separately for each tissue and species. Significant differences (p < .05) are indicated by different letters. There were no significant differences (p > .05) observed in the amino acid concentrations in the E. mactroides mantle and adductor muscle or the A. mactroides mantle and gills.

2014; Matsushima and Kado, 1982), which maintain their bodily fluid osmolality minimally higher to guarantee survival in environments with very low salinities. Consequently, the effects of the variation in the hemolymph osmotic concentration could be visualized by the decrease in the mantle, adductor muscle, and gill water content in this freshwater species (Fig. 3a-c). These observations corroborate several 8

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Fig. 8. Estimation of ancestral states of some representatives of the Bivalvia class, by maximum likelihood analysis, for hemolymph osmolality (mOsm/kg H₂O; left panel) and habitat osmolality (%₀; right panel). Both traits are positively correlated (PGLS, F = 5133.4, p < .0001, ɑ = 64.4).

Fig. 9. Estimation of ancestral states for some representatives of the class Bivalvia, by maximum likelihood analysis, for the [Na+] (mM; left panel) and [Cl−] (mM; right panel). Both traits are positively correlated (PGLS, F = 314.9, p < .0001; ɑ = 0.83).

comparative approach revealed that closely related species present similar [K+]. This data indicate possible conservation of this cation in the hemolymph fluid of this group, regardless of the habitat salinity. In contrast, the other evaluated ions (Na+, Cl−, Mg2+ and Ca2+) do not appear to be phylogenetically structured. Although there was no significant phylogenetic signal for the [Na+], we observed a tendency in certain clades to present higher hemolymph values. On the other hand, considering the hemolymph fraction that corresponded to [Na+] and [Cl−] (approximately 90%), the analyses between both physiological traits reveal that there is no phylogenetic structuring between them. Thus, when in natural salinities, species that have lower [Na+] concomitantly present lower [Cl−] (Fig. 9). Osmolality is an important environmental factor that imposes great selection pressure on the evolution of life. As a result, all existing cells have, in a variable way, mechanisms of cell volume regulation—activated when in osmotic shock situations—which control the concentration of the cytoplasmic compartment for which cellular metabolism is optimized (Chamberlin and Strange, 1989; Kultz, 2000; Pierce, 1982). In this sense, the IIR capacity constitutes a fundamental characteristic in osmoconformer organisms that tend to occupy variable osmotic niches. Based on the necessary adjustments on the osmotic effectors (i.e., free ions and amino acids), the RVI and RVD strategies seek to compensate for and control the adverse effects of salinity fluctuations on cells. Although the scientific literature reports the preferential use of certain osmolytes by organisms from distinct niches (see Burg and Ferraris, 2008; Kinne, 1993; Yancey, 2005), such conclusions are still far from the comparative context, and thus they can only be

considered rules once comparative information is reported for a limited number of species in view of the diversity of each taxon. Pierce (1982) notes that the relative contribution of each type of solute tends to vary according to species and cell type as well as the osmotic stress amplitude and duration of osmotic stress. Additionally, different tissues have distinct responses to salinity variations (Carregosa et al., 2014; Deaton, 1994; Pierce Jr, 1971; Ruiz and Souza, 2008). Here, we identified that, when exposed to hyperosmotic shock, the C. largillierti mantle and gills exhibited increased [Na+], [K+], and [Cl−], and the adductor muscle showed elevated [K+] and [Cl−] (Fig. 4a-c). Investigations reported that freshwater bivalve mollusks (cells from freshwater invertebrates) mainly use ions as intracellular effector osmolytes (Potts, 1958; Murphy and Dietz, 1976; Gainey and Greenberg, 1977; Deaton and Greenberg, 1991; Dietz et al., 1997; Pierce, 1982). Interestingly, the investigated freshwater species also showed a FAA increase in the evaluated tissues when they were exposed to higher salinities (Fig. 7a). Although they are the class of effectors that most varies in C. largillierti, their contribution as effective osmolytes is much smaller when compared with the inorganic ions (mM). However, this observation is surprising because freshwater organisms, although not naturally exposed to hyperosmotic conditions, may accumulate FAA in response to increased salinity (Matsushima et al., 1987). Physiologically, some studies indicated that freshwater bivalves possess the ability to increase some amino acids under hyperosmosis (Hosoi et al., 2008). Some authors also suggest that the Corbiculidae family has a relatively short fossil history, so it is generally accepted that Corbicula spp. are very recent immigrants in freshwater (Keen and Casey, 1969; 9

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McMahon, 1983). Thus, such an evolutionary background seems to explain the FAA accumulation capacity when subjected to hyperosmotic shock (Matsushima et al., 1987), mainly for C. largillierti. In E. mactroides, the tissues expressed distinct patterns with regards to osmolyte recruitment in the view of volume regulation (Fig. 5a-c). In contrast to the hypoosmotic profile, the estuarine bivalve specimens submitted to hyperosmotic salinity presented higher [Na+], as well as a slight tendency to increase the [Cl−] content in those tissues in constant interface with the external environment (gills and mantle). Additionally, the [K+] remained higher in the mantle after the end of the acute exposure to high salinity (14%₀). On the other hand, the adductor muscle, which tends to be more anatomically protected in the animal, remained independent to any water change and also exhibited no variations in osmolyte concentrations. It is thought that lower permeability to the osmotic variations particularly in this tissue ensures the said pattern observed even at different osmotic concentrations. Concerning the participation of organic osmolytes in E. mactroides IIR, in general they were not apparently widely recruited for the maintenance of tissue equilibrium for either osmotic shock. Thus, we conclude that to maintain the cellular volume when in the hyper- or hypoosmotic medium, only the inorganic osmolytes remain at distinct concentrations according to the salinity experienced by the mantle and gills (Fig. 7b). Although most data indicate that osmoconformer cells utilize a pool of intracellular FAA as the substantial solute source for osmotic equilibrium, there is evidence that inorganic osmolytes are also used as effectors in these cells (Pierce, 1982). A classic profile expected for species that inhabit the marine niche is based on the use of organic osmolytes to maintain the osmotic cellular concentration (Yancey, 2005). Here, A. mactroides exposure to hyperosmotic shock caused a significant increase in the FAA concentration only in the adductor muscle, although there was also a slight tendency in the mantle and gills for higher FAA concentrations (Fig. 7c). In addition to these effects, the aforementioned treatment also increased the mantle, adductor muscle, and gill [Na+] (Fig. 6a-c), data that are more representative than the variations observed in the FAA content. These observations were the opposite after the animals were submitted to hypoosmotic conditions. Regarding these findings, Carregosa et al. (2014) note that marine organisms reach osmotic equilibrium mainly with Na+. Berger and Kharazova (1997) further recognize the key role played by [Na+] in the osmotic regulation of organisms exposed to changes in external salinity. Based on the physiological patterns experimentally verified in C. largillierti (purple Asian cockle), E. mactroides (lagoon cockle), and A. mactroides (white clam) we demonstrated that these species were osmoconformers under the tested salinities. Additionally, these bivalve species used more inorganic osmolytes than organic molecules as IIR effectors in the evaluated tissues, albeit in different ways. After exposure to the experimental conditions, C. largillierti was prone to the use of inorganic osmolytes (Na+, K+ and Cl−), and to a lesser extent organic ones (i.e., FAA). On the other hand, E. mactroides used only Na+ and K+, while A. mactroides specifically recruited Na+ ions as well as very low amounts of FAA as effectors for its regulation. This finding expands the classic vision about the preferential use of osmolyte effectors by aquatic invertebrates in the physiological process of osmoregulation. From our comparative analyses conducted with Bivalvia representatives and inhabitants of the same studied osmotic niches, we revealed that environmental salinity plays an important role in the hemolymph osmolality, as well as in concentration of the main ions that comprise the extracellular fluid (Na+, Mg2+, Ca2+, and Cl-). [K+] variability, specifically, follows a hierarchical fashion owing the significant phylogenetic signal, revealing the importance of the osmotic environment and shared ancestry in the evolution of ionic regulation. Finally, the observations reported here in relation to the experienced osmotic scenarios (hypo- and/or hyperosmotic) by the bivalve species, as well as the general verified patterns, enable the reporting and

inference of physiological aspects—due to their comparative character—until then not described for the taxon. Declaration of Competing Interest The authors attest that there is no conflict of interest. Acknowledgments We thank Dr. Ricardo Berteaux Robaldo (IB-UFPel), Dr. Leonir André Colling (IO-FURG) and MSc. Fernanda Chaves Lopes for their collaboration in the effort to collect freshwater, estuarine and marine specimens, as well as the Laboratory of Phytoplankton and Marine Microorganisms (IO - FURG) for the microalgae donation used to maintain the bivalves of this study. We are also grateful to Dr. Patrícia Costa, Dr. Ana Laura Escarrone and Dr. Micheli Castro for the contributions together with flame photometry and amino acid analyzes. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001. Samuel C. Faria is a postdoc fellow from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2017/05310-9 and #2018/21670-8). Marta M. Souza is a research fellow from the Brazilian CNPq (Proc. #311806/2017-1). References Allen, J.A., Garrett, M.R., 1972. Studies on taurine in the euryhaline bivalve Mya arenaria. Comp. Biochem. Physiol. A 41, 307–317. https://doi.org/10.1016/0300-9629(72) 90062-X. Amado, E.M., Freire, C.A., Souza, M.M., 2006. Osmoregulation and tissue water regulation in the freshwater red crab Dilocarcinus pagei (Crustacea, Decapoda), and the effect of waterborne inorganic lead. Aquat. Toxicol. 79, 1–8. https://doi.org/10. 1016/j.aquatox.2006.04.003. Amado, E.M., Vidolin, D., Freire, C.A., Souza, M.M., 2011. Distinct patterns of water and osmolyte control between intertidal (Bunodosoma caissarum) and subtidal (Anemonia sargassensis) sea anemones. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 158, 542–551. https://doi.org/10.1016/j.cbpa.2010.12.019. Babarro, J.M.F., Fernández Reiriz, M.J., Labarta, U., Garrido, J.L., 2011. Variability of the total free amino acid (TFAA) pool in Mytilus galloprovincialis cultured on a raft system. Effect of body size. Aquac. Nutr. 17, e448–e458. https://doi.org/10.1111/j.13652095.2010.00781.x. Baginski, R., Pierce Jr., S., 1978. A comparison of amino acid accumulation during high salinity adaptation with anaerobic metabolism in the ribbed mussel, Modiolus dernissus dernissus. J. Exp. Zool. 203, 419–428. Beadle, L.C., 1957. Comparative physiology: osmotic and ionic regulation in aquatic animals. Annu. Rev. Physiol. 19, 329–358. Berger, V.J., Kharazova, A.D., 1997. Mechanisms of salinity adaptations in marine molluscs. Hydrobiologia 355, 115–126. Berger, V.Ya., Khlebovich, V.V., Kovaleva, N.M., Natochin, Yu.V., 1978. The changes of ionic composition and cell volume during adaptation of molluscs (Littorina) to lowered salinity. Comp. Biochem. Physiol. A 60, 447–452. https://doi.org/10.1016/ 0300-9629(78)90015-4. Burg, M.B., Ferraris, J.D., 2008. Intracellular organic osmolytes: function and regulation. J. Biol. Chem. 283, 7309–7313. https://doi.org/10.1074/jbc.R700042200. Burg, M.B., Ferraris, J.D., Dmitrieva, N.I., 2007. Cellular response to hyperosmotic stresses. Physiol. Rev. 87, 1441–1474. https://doi.org/10.1152/physrev.00056. 2006. Butler, M., King, A., 2004. Phylogenetic comparative analysis: a modeling approach for adaptive evolution. Am. Nat. 164, 683–695. Byrne, R.A., Dietz, T.H., 2006. onic and acid-base consequences of exposure to increased salinity in the zebra mussel, Dreissena polymorpha. Biol. Bull. 211 (1), 66–75. https://doi.org/10.2307/4134579. Carregosa, V., Figueira, E., Gil, A.M., Pereira, S., Pinto, J., Soares, A.M.V.M., Freitas, R., 2014. Tolerance of Venerupis philippinarum to salinity: osmotic and metabolic aspects. Comp. Biochem. Physiol. A 171, 36–43. https://doi.org/10.1016/j.cbpa.2014.02. 009. Carvalho, Y., Romano, L., Poersch, L., 2015. Effect of low salinity on the yellow clam Mesodesma mactroides. Braz. J. Biol. 75, 8–12. https://doi.org/10.1590/1519-6984. 03213. Chamberlin, M.E., Strange, K., 1989. Anisosmotic cell volume regulation: a comparative view. Am. J. Phys. Cell Phys. 257, C159–C173. https://doi.org/10.1152/ajpcell. 1989.257.2.C159. Costa, C.J., Pritchard, A.W., 1978. The response of Mytilus edulis to short duration hypoosmotic stress. Comp. Biochem. Physiol. A 61, 149–155. https://doi.org/10.1016/ 0300-9629(78)90292-X. Coughlan, B.M., Moroney, G.A., Pelt, F.N.A.M.v., O'Brien, N.M., Davenport, J., O'Halloran, J., 2009. The effects of salinity on the Manila clam (Ruditapes philippinarum) using the neutral red retention assay with adapted physiological saline

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