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Anaerobiosis and metabolic plasticity of Pinna nobilis: Biochemical and ecological features
Biochemical Systematics and Ecology 56 (2014) 138e143
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Anaerobiosis and metabolic plasticity of Pinna nobilis: Biochemical and ecological features a, D. Barreca a, *, S. Giacobbe b, E. Bellocco a G. Lagana a b
di Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy Dipartimento di Scienze Chimiche, Universita di Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy Dipartimento di Scienze Ambientali e Biologiche, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2013 Accepted 31 May 2014 Available online
Changes in the energetic metabolism were studied in the fan mussel Pinna nobilis L. exposed to environmental and anthropic stress. The high polymorphism of enzymes suggests an adaptation of the fan mussel to environmental variability peculiar of transitional waters with respect to the same species living in exposed coastal sea. The electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH isozymes. Moreover, in all the analyzed tissues and organs, MDH activity was greater than the LDH one. Metabolic plasticity of the fan mussel is further highlighted by octopine dehydrogenase and superoxide dismutase electrophoretic patterns, showing the presence of many isoforms. These evidences are also confirmed by spectroscopic determinations of alanopine, tauropine, strombine and octopine dehydrogenase activity characterized by a specific trend due to environmental variability. Specific variations in anaerobic capacity of P. nobilis L. are discussed in relation to their distribution according to the marine-brackish gradient. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Pinna nobilis Environmental adaptation Energetic metabolism Opine dehydrogenase Superoxide dismutase
1. Introduction Marine organisms modulate their metabolic pathways to counteract environmental factors such as temperature, salinity, oxygen or nutrient availability, maximizing benefits during favorable conditions and allowing survival during critical stages. For example, in many intertidal and infaunal invertebrates, the lack of a constant oxygen supply results in some spectacular tolerances (De Zwaan, 1977; Hochachka and Somero, 2002). These features are similar to some opportunistic species that can dominate a subtidal environment following a dystrophic crisis (Sato, 2006). Bivalves have evolved diverse and highly specialised strategies for surviving in hypoxic episodes including pathways that are efficient both in terms of ATP production, and in minimising Hþ and toxic end product accumulation (Hochachka and Somero, 2002). Under these circumstances, glycogen is metabolized to pyruvate and the cytosolic NADH/NADþ redox ratio is balanced by the reduction of pyruvate to lactate. Alternatively, NADþ can be recycled more efficiently by coupling an aminoacid to pyruvate, with formation of opines such as €de, 1983; Ga €de and Grieshaber, 1986). Stress factors induce alterations both alanopine, tauropine, octopine, and strombine (Ga in the amount of enzymes and in the production of isoforms possessing altered kinetic properties or post-translational modifications. In particular, since the estuarine and intertidal species are submitted naturally to multiple forms of daily stress, they display a wide range of metabolic adaptations, involving peculiar biochemical features (Storey and Storey, 1990;
* Corresponding author. Tel.: þ39 090 6765187; fax: þ39 090 6765186. E-mail address: [email protected] (D. Barreca). http://dx.doi.org/10.1016/j.bse.2014.05.020 0305-1978/© 2014 Elsevier Ltd. All rights reserved.
et al. / Biochemical Systematics and Ecology 56 (2014) 138e143 G. Lagana
139
lez-Wangüemerta et al., 2009). These adaptations of estuarine and Hochachka, 1997; Hochachka and Somero, 2002; Gonza intertidal bivalves to environmental variability have been poorly investigated in marine subtidal species, although they are €de and Grieshaber, 1986; frequently submitted to several types of stress causing short-term anaerobiosis (De Zwaan, 1977; Ga Fields and Storey, 1987). The Mediterranean endemic Pinna nobilis L., is one of the largest bivalves in the world. The “noble fan shell” lives partially buried in the soft bottom, with the broad posterior part of the shell that is elevated well above the sea floor. Fan shells are considered ecosystem engineers that remarkable increase habitat complexity, host epibiontic assemblages (Corriero and Pronzato, 1987; Cosentino and Giacobbe, 2008; Rabaoui et al., 2009) and locally influence oxygen and nutrient fluxes (Hewitt et al., 2006). Noble fan shell beds mostly occur from a maximum depth of 60 m in wave exposed areas up to 0.5 m in coastal lagoons (Zavodnik et al., 1991). In this respect, fan mussels might represent a profitable case-study to investigate metabolic adaptation of subtidal bivalves to environmental stress in oligotrophic shallow waters of sub-tropical areas (Butler et al., 1993). In this study, enzymatic markers and isozymes electrophoretic patterns of P. nobilis have been investigated in samples that are representative of a marine-brackish water gradient, to evaluate energetic metabolism and anaerobic adaptation in a long-lived moderately eurivalent species. 2. Methods 2.1. Sampling sites The specimens of P. nobilis were collected in the Straits of Messina area (central Mediterranean) between May and June of 2011, from two nearby marine and brackish-water sites; the respective populations, that are not spatially separated, in agreement with Butler et al. (1993), might belong to the same metapopulation. The marine sites included a wave exposed seafloor (St.1) and a more sheltered area (St.2), both at 7 m depth. The brackish water sites were located inside the channel that leads to the Faro lake (St.3) and in the inner part of the same lake (St.4), both at 1 m depth. In the Straits of Messina, the marine environment is characterized by tidal currents having a maximum speed of 3 m s1, with a period of 6 ¼ h. Such hydrological constraint leads to the upwelling of “Levantine Intermediate Water” that is colder, more salty and more nutrient-rich compared to the surface waters of Atlantic origin (Azzaro et al., 2007). This increase of primary production supports high density of benthic suspension feeders due to the water turbulences, oxygen availability and catabolytes clearing (Leonardi et al., 2009). The Faro lake is a meromictic basin, reaching a maximum depth of 29 m, characterized by anoxic and sulfidic waters, generally below 15 m in depth. Surface waters are mesotrophic, with a predominant heterotrophic biomass in the particulate matter and poorly oxygenated water can spread towards the surface in autumn. Exceptional seawater inflows cause strong alteration of the anoxic layer and temporary diseases on all aerobic organisms (Leonardi et al., 2009). 2.2. Sampling and sample treatment Adult specimens having a comparable shell size were collected manually in each site and transported alive, within 10 min, into the laboratory by means of a thermic bag. Once sacrificed, they were measured according to García-March and Ferrer (1995) and their biomass was evaluated as shell-free wet weight. Soft tissues were weighted, dissected, and frozen at 20 C until analysis. Adductor muscle, epatopancreas, gills and mantle were homogenized (1:4 w/v), using an Ultra Turrax tissue grinder, at 4 C in 0.01 M potassium phosphate buffer (pH 7) containing 0.5 mM phenylmethyl sulfonyl fluoride (PMSF) and 1 mM b-mercaptoethanol. The homogenate was centrifuged for 15 min at 12 000 rpm at 4 C. The supernatant was submitted to enzyme assays, protein determination and electrophoretic analysis. 2.3. Total protein and enzyme activities determinations Total protein content was determined according to Bradford (1976). The dehydrogenase activities were analyzed at et al. 25 ± 0.1 C following the changes of absorbance at 340 nm. LDH and MDH activity was assayed according to Lagana (2007). Alanopine (ADH), tauropine (TDH), strombine (SDH) and octopine (ODH) dehydrogenase activity was performed according to Baldwin et al. (1992). All the analyses were carried out in triplicate. One enzyme unit was defined as the amount catalyzing the production of 1.0 mM NAD þ per min at 25 C. In each experiment the values are the means ± standard deviation of three independent experiments (n ¼ 3). Statistical comparisons of the results were performed by one-way ANOVA. Significant differences (p < 0.05) between the tested samples were analyzed using Turkey’s test. 2.4. Isozymes determinations et al. (2007). LDH staining soVertical electrophoresis was performed on 7.5% polyacrylamide gel according to Lagana lutions consisted of 60 mM lithium lactate, 0.336 mM nicotinamide adenine dinucleotide (NADþ), 0.168 mM Nitro-blue tetrazolium (NBT), 0.056 mM phenazine methosulfate (PMS) in 0.1 M phosphate buffer (pH 7.0). MDH and ODH staining solution contained the same elements, but litium lactate was replaced with malate or octopine respectively. Staining of SOD was performed immediately after the achievement of electrophoresis using the nitrobluetetrazolium (NBT) photochemical method of Beauchamp and Fridovich (1971).
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Table 1 Protein (mg/g) content in the organs and tissues of the collected specimens. St.1 Epatopancreas Gills Adductor muscle Mantle
St.2
10.62 1.71 23.50 1.16
± ± ± ±
0.53a 0.38a 0.85a 0.3a
18.35 6.3 21.05 1.85
St.3 ± ± ± ±
1.26b 0.41b 1.39a 0.25a
15.50 6.00 27.08 3.75
St.4 ± ± ± ±
0.65c 0.24b 1.83b 0.46b
13.00 12.80 23.70 4.20
± ± ± ±
0.74d 0.95c 1.68a 0.72b
Different letters indicate significant difference (mean ± SD; n 3; p < 0.05).
3. Results The examined specimens ranged in size between 34 and 37 cm, with a biomass of 42 ge45 g. The marine specimens hosted the endo-commensalistic shrimp Pontonia pynnophylax, which was absent in the specimens collected in the lake and channel. The specimens collected in St.2, St.3 and St.4 showed hypertrophic gills and particularly those collected in the last station. Protein amount in the adductor muscle, epatopancreas, gills and mantle is summarized in Table 1. As can be seen epatopancreas and adductor muscles showed the highest protein content. Our survey of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) is summarized in Tables 2 and 3. It reveals marked differences both in the enzymatic activities and in the isoforms expression patterns. In all the analyzed tissues, MDH activity was usually much higher than the LDH one. The adductor muscle showed the highest level of MDH (4.73e6.19 U/g), followed by epatopancreas (0.86e4.43 U/g). Moreover samples collected in brachish habitat had higher levels of enzymes than those collected in the waves exposed station. Lactate dehydrogenase activity was found in dosable amounts only in the adductor muscle (0.33e0.65 U/g) and in the epatopancreas (0.74e0.98 U/g), while it was undetectable in the gills and in the mantle. In a different way from MDH, the samples collected in the waves exposed St.1 showed the highest level of LDH. The analyzed specimens had many opine dehydrogenases, whose function is similar to that of LDH. Fig. 1 shows the enzymatic activity of the tested opine dehydrogenases in the adductor muscle. These dehydrogenases can oxidize NADH produced by glycolysis and promote glycolysis to yield more ATP for physiological functioning under anoxia. The enzyme octopine dehydrogenase (ODH) showed the same tissue distribution evidenced by LDH, although the U/g were remarkably higher in the former instead of the latter. The adductor muscle also showed a conspicuous amount (U/g) of alanopine, tauropine and strombine dehydrogenase (Fig. 1). These enzymes were present only in trace in epatopancreas (data not shown). ODH is the major opine dehydrogenase in the adductor muscle, while ADH, SDH and TDH show almost the same activity. As can be seen from the graph, the U/g of ODH increased following the marine-brackish gradient (from specimens collected in St.1 to the ones collected in St.4). On the contrary, the U/g of the other analyzed opine dehydrogenases decreased following the same gradient (Fig. 1). The electrophoretic profiles of lactate, octopine and malate dehydrogenases are characterized by the presence of multiple isoenzymes. LDH isoenzymes are due to the A and B genes expression, resulting in the formation of homotetramers and one or more heterotetramers. The electrophoretic pattern generally showed the presence of three anodic bands only at the level of the adductor muscle and epatopancreas with little intensity differences between them (Fig. 2). In the epatopancreas of samples collected in St.4 no band was detected. The adductor muscle showed the presence of three bands in the specimens collected in St.1, St.2 and St.3, while only two bands were present in the samples collected in St.4 (Fig. 2). As in the case of LDH, octopine dehydrogenase pattern showed the presence of many bands only in the adductor muscle and in the epatopancreas. All the monitored isoforms were anodic, showing good eloctrophoretic mobility (Fig. 2). Only the samples collected in St.4 and St.1 showed significant bands at the level of the epatopancreas, consisting in two or three isozymes respectively (Rf between 0.63 and 0.74). Moreover the bands intensity was much more noticeable in the former than in the latter. In the adductor muscle there was the presence of at least three bands with good anodic electrophoretic mobility in all the samples. Moreover in the samples collected in St.4 there was a further isozyme, more anodic, with a Rf of 0.74. MDH activity is evident in all the samples analyzed, with clearly cathodic bands. Gills, epatopancreas and adductor muscle of the specimens collected in St.3 showed further isozymes with intermediate electrophoretic mobility, as well as the adductor muscle of the specimens collected in St.4. Seven isozymes were present in the gills of P. nobilis collected in St. 3, four similar to the ones present in the other samples and three characteristic of this habitat. A lower number of isozymes were present in the epatopancreas of the analyzed specimens with respect to the
Table 2 MDH activity (U/g) present in the organs and tissues of the collected specimens.
Epatopancreas Gills Adductor muscle Mantle
St.1 (wave exposed)
St.2 (sheltered)
St.3 (channel)
St.4 (lake)
MDH U/g
MDH U/g
MDH U/g
MDH U/g
0.86 0.35 4.73 0.52
± ± ± ±
0.03a 0.05a 0.12a 0.05a
3.21 1.40* 5.31 1.68
Different letters indicate significant difference (mean ± SD; n 3; p < 0.05). * hypertrophic; ** highly hypertrophic.
± ± ± ±
0.08b 0.10b 0.09b 0.07b
4.43 2.62* 6.19 1.09
± ± ± ±
0.12c 0.08c 0.09c 0.09c
1.58 1.65** 5.48 2.61
± ± ± ±
0.11d 0.07d 0.11b 0.1d
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Table 3 LDH activity (U/g) present in the organs and tissues of the collected specimens.
Epatopancreas Adductor muscle
St.1 (wave exposed)
St.2 (sheltered)
St.3 (channel)
St.4 (lake)
LDH U/g
LDH U/g
LDH U/g
LDH U/g
0.98 ± 0.06a 0.65 ± 0.07a
0.80 ± 0.08b 0.33 ± 0.05b
0.74 ± 0.04b 0.35 ± 0.05b
e 0.36 ± 0.07b
Different letters indicate significant difference (mean ± SD; n 3; p < 0.05). In gills and mantle we do not find detectable LDH activity.
gills. The mantle showed the presence of three main isozymes common to all the habitats, except for the samples collected in St.1 that had two bands corresponding to the more cathodic ones present in all the other samples. The electrophoretic pattern of the adductor muscle showed the presence of three cathodic isozymes (Rf of 0.09, 0.11 and 0.14). Moreover, the samples collected in St.3 and St.4 showed further less cathodic bands than the ones above mentioned, one coincident in the two habitats and another characteristic of the latter. The necessity to overcome oxidative injuries was highlighted by SOD electrophoretic pattern. Almost all the samples evidenced at least two SOD isozymes. In the gills were detected two bands common to all the habitats, except for those collected in St.4 that presented only one isoform. The specimens collected in St.2 and St.3 also showed a third band with higher mobility. Two SOD isozymes were present in the epatopancreas of the samples collected in St.1 and St.4, while those collected in St.2 and St.3 showed three bands. The mantle also showed the same electrophoretic pattern evidenced for the epatopancreas. The electrophoretic pattern of the adductor muscle showed two corresponding isozymes in the samples collected in St.1, St.2 and St.3; in this latter one was also present a third band with higher mobility. Only a cathodic band was present in the samples collected in St 4.
4. Discussion The marine intertidal and infaunal invertebrates give rise to biochemical changes and/or adaptive responses characteristic of the species and of the marine-brackish gradient. Assessment of biochemical features is essential to understand the influences of environmental changes on organism survival and to predict their interaction with the habitat. In all the tissues the expression of three separate gene loci (Mdh-M, Mdh-A and Mdh-B) of MDH was found. The presence of MDH-AB heterodimers, in almost all samples, indicates the simultaneous synthesis of both MDH-A and B subunits in many differentiated cells. Although the electrophoretic patterns exhibited essentially similar gene expression in the four tissues, they showed some habitat-specific isoforms. The most observed differences concerned the electrophoretic mobility and the relative intensity of the detected isozymes. In particular, isozyme electrophoretic patterns showed the presence of an anaerobic LDH isoenzyme and MDH isozymes (cytosolic and mitochondrial isoforms). The presence of opine dehydrogenases gives evidence of a remarkable metabolic plasticity and adaptability to environmental changes, according to literature data for estuarine and coastal bivalves (Hochachka and Somero, 2002). This is probably due to the energy need to overcome the changes associated with marine-brackish gradient. In fact opine dehydrogenase are able to produce more energy than the one that can be provided by the LDH route alone. The distribution of pyruvate reductases is strongly aligned with the ability to tolerate environmental hypoxia or to perform muscular work. Evidence from enzyme kinetics indicates that the ODH pathway is €de, 1983). Thus, ODH probably able to realize a higher rate of energy production than either the SDH or ADH pathways (Ga correlates with functional hypoxia during muscular activity, whereas SDH and LDH correlate with periods of environmental hypoxia. According to this data the ODH activity in adductor muscle increases following the marine-brackish gradient, while the one of ADH, SDH and TDH decreases following the same gradient. Moreover, anaerobic pathways are not all equivalent in terms of energy production based upon maximum rates for ATP output (lactate > octopine > alanopine ¼ strombine). For this reason, specimens utilizing the octopine (than alanopine) pathway will increase energy flow rapidly, developing a major ability to counteract environmental variations. This metabolic condition might conceivably facilitate a higher glycolytic rate and the production of a substrate (arginine) for the formation of octopine. In accordance with other mollusks that use a
Fig. 1. Opine dehydrogenases. Activities of ODH, ADH, SDH and TDH in the adductor muscle.
et al. / Biochemical Systematics and Ecology 56 (2014) 138e143 G. Lagana
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Fig. 2. LDH and ODH isozymes. Electrophoretic pattern of LDH in epatopancreas and adductor muscle. The extracts were submitted to electrophoretic analysis and specifically staining for LDH or ODH activity.
variety of fermentation pathways, the absence of opine dehydrogenases in some tissues could be due to the utilization of the €rtner et al., 1984; Grieshaber et al., 1994). propionic acid pathway (Po The high ratio between MDH/LDH could be due to the ability of P. nobilis to turn on anaerobic metabolism as a consequence of environmental or anthropogenic stresses. High fluctuations of the thermo-aline regime, which especially affect the marineecontinental transition, explain the higher variability of enzymatic pattern, showed by the channel specimens. Recent studies have revealed that environmental temperature significantly affects both the total metabolic rate and metabolic € rtner, regulation in aquatic ectotherms, eliciting transition to anaerobiosis even in fully oxygenated waters (Sokolova and Po 2003). This experimental evidence is further supported by the electrophoretic patterns of SOD isozymes. Those showed the higher variability in the specimens collected in St.2 and St.3. In fact, it is well known that reactive oxygen species (ROS) are produced in cells subjected to environmental stresses such as exposure to intense light, UV radiations, metals, xenobiotics, thermal shock and change in salinity. Therefore, the induction of antioxidant enzymes like SOD is an important protective mechanism to minimize cell oxidation. In conclusion, the differences in the ability to withstand prolonged anoxia or rapid changes in environmental conditions (e.g. marine-brackish gradient) are correlated to a certain extent with the ecological peculiarities of the studied species and specifically with their biochemical pattern of enzyme expression. Acknowledgments The authors would like to thank William Fenton for his contribution in linguistic revision of the text. References Azzaro, F., Decembrini, F., Raffa, F., Crisafi, E., 2007. Seasonal variability of phytoplankton fluorescence in relation to the Straits of Messina (Sicily) tidal upwelling. Ocean. Sci. 3, 451e460. Baldwin, J., Wells, R.M.G., Low, M., Ryder, J.M., 1992. Tauropine and D-lactate as metabolic stress indicators during transport and storage of live Paua, (New Zealand Abalone) (Haliotis iris). J. Food Sci. 57, 280e282. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem 44, 276e287. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72, 248e254. Butler, A., Vicente, N., Gaulejac, B., 1993. Ecology of the pterioid bivalves Pinna bicolor Gmelin and Pinna nobilis L. Mar. Life 3, 37e45. Corriero, G., Pronzato, R., 1987. Epibiontic sponges on the bivalve Pinna nobilis. Mar. Ecol. Prog. Ser. 35, 75e82. Cosentino, A., Giacobbe, S., 2008. Aspects of epizoobiontic mollusc assemblages on Pinna shells. II. Does the Mediterranean P. nobilis represent an isle of biodiversity? Cah. Biol. Mar. 49, 161e173. De Zwaan, A., 1977. Anaerobic energy metabolism in bivalve molluscs. Oceanogr. Mar. Biol. A. Rev. 15, 103e187. Fields, J.H.A., Storey, K.B., 1987. Tissue-specific alanopine dehydrogenase from gill and strombine dehydrogenase from the foot muscle of the cherrystone clam Mercenaria rnercenana (Linn.). J. Exp. Mar. Biol. Ecol. 105, 175e185. €de, G., 1983. Energy metabolism of arthropods and mollusks during environmental and functional anaerobiosis. J. Exp. Zool. 228, 415e429. Ga €de, G., Grieshaber, M.K., 1986. Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates. Comp. Biochem. Physiol. 83, Ga 255e272. n dela equacio n de De Gaulejac y Vicente (1990). Bol. Inst. Esp. Oceanogr. 11, García-March, J.R., Ferrer, J., 1995. Biometría de Pinna nobilis L., 1758: una revisio 175e181. rez-Ruzafac, A., 2009. Phosphoglucose isomerase variability of Cerastoderma glaucum as a model Gonz alez-Wangüemerta, M., C anovasb, F., Marcosc, C., Pe for testing the influence of environmental conditions and dispersal patterns through quantitative ecology approaches. Biochem. Syst. Ecol. 37, 325e333.
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€ rtner, H.O., 1994. Physiological and metabolic responses to hypoxia in invertebrates. Reviews of Physiology. Grieshaber, M.K., Hardewig, I., Kreutzer, U., Po Biochem. Pharmacol. 125, 43e147. Hewitt, J., Thrush, S., Gibbs, M., Lohrer, D., Norkko, A., 2006. Indirect effects of Atrina zalandica on water column nitrogen and oxygen fluxes: the role of benthic macrofauna and microphytes. J. Exp. Mar. Biol. Ecol. 330, 261e273. Hochachka, P.W., 1997. Oxygen-a key regulatory metabolite in metabolic defense against hypoxia. Am. Zool. 37, 595e603. Hochachka, P.W., Somero, G.N., 2002. Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York. Lagan a, G., Giacobbe, S., Bellocco, E., Mannucci, C., Galtieri, A., Ficarra, S., Kotyk, A., Leuzzi, U., 2007. Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): effects of temperature and urea. J. Exp. Mar. Biol. Ecol. 349, 27e34. Leonardi, M., Azzaro, F., Azzaro, M., Caruso, G., Mancuso, M., Monticelli, L.S., Maimone, G., La Ferla, R., Raffa, F., Zaccone, R., 2009. A multidisciplinary study oft he Capo Peloro brackish area (Messina, Italy): characterization of the trophic conditions, microbial abundances and activities. Mar. Ecol. 30, 33e42. € rtner, H.O., Heisler, N., Grieshaber, M.K., 1984. Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by Po anaerobic metabolism. J. Comp. Physiol. B 155, 1e12. Rabaoui, L., Tlig-Zouari, S., Cosentino, A., Ben Hassine, O.K., 2009. Associated fauna of the fan shell Pinna nobilis (Mollusca:Bivalvia) in the northern and eastern Tunisian coasts. Sci. Mar. 73, 129e141. Sato, S., 2006. Drastic change of bivalves and gastropods caused by the huge reclamation projects in Japan and Korea. Plankton Benthos Res. 1, 123e137. € rtner, H.-O., 2003. Metabolic plasticity and critical temperatures for aerobic scope in a eurythermal marine invertebrate (Littorina saxatilis, Sokolova, I.M., Po Gastropoda: Littorinidae) from different latitudes. J. Exp. Biol. 206, 195e207. Storey, K.B., Storey, J.M., 1990. Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation, and estivation. Q. Rev. Biol. 65, 145e193. Zavodnik, D., Hrs-Brenko, M., Legac, M., 1991. Synopsis on the fan shell P. nobilis L. in the eastern Adriatic sea. In: Boudouresque, C.F., Avon, M., Gravez, V. ces Marines a Prote ger en Me diterrane e. GIS Posidonie, Marseille, pp. 169e178. (Eds.), Les Espe
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Anaerobiosis and metabolic plasticity of Pinna nobilis: Biochemical and ecological features a, D. Barreca a, *, S. Giacobbe b, E. Bellocco a G. Lagana a b
di Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy Dipartimento di Scienze Chimiche, Universita di Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy Dipartimento di Scienze Ambientali e Biologiche, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2013 Accepted 31 May 2014 Available online
Changes in the energetic metabolism were studied in the fan mussel Pinna nobilis L. exposed to environmental and anthropic stress. The high polymorphism of enzymes suggests an adaptation of the fan mussel to environmental variability peculiar of transitional waters with respect to the same species living in exposed coastal sea. The electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH isozymes. Moreover, in all the analyzed tissues and organs, MDH activity was greater than the LDH one. Metabolic plasticity of the fan mussel is further highlighted by octopine dehydrogenase and superoxide dismutase electrophoretic patterns, showing the presence of many isoforms. These evidences are also confirmed by spectroscopic determinations of alanopine, tauropine, strombine and octopine dehydrogenase activity characterized by a specific trend due to environmental variability. Specific variations in anaerobic capacity of P. nobilis L. are discussed in relation to their distribution according to the marine-brackish gradient. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Pinna nobilis Environmental adaptation Energetic metabolism Opine dehydrogenase Superoxide dismutase
1. Introduction Marine organisms modulate their metabolic pathways to counteract environmental factors such as temperature, salinity, oxygen or nutrient availability, maximizing benefits during favorable conditions and allowing survival during critical stages. For example, in many intertidal and infaunal invertebrates, the lack of a constant oxygen supply results in some spectacular tolerances (De Zwaan, 1977; Hochachka and Somero, 2002). These features are similar to some opportunistic species that can dominate a subtidal environment following a dystrophic crisis (Sato, 2006). Bivalves have evolved diverse and highly specialised strategies for surviving in hypoxic episodes including pathways that are efficient both in terms of ATP production, and in minimising Hþ and toxic end product accumulation (Hochachka and Somero, 2002). Under these circumstances, glycogen is metabolized to pyruvate and the cytosolic NADH/NADþ redox ratio is balanced by the reduction of pyruvate to lactate. Alternatively, NADþ can be recycled more efficiently by coupling an aminoacid to pyruvate, with formation of opines such as €de, 1983; Ga €de and Grieshaber, 1986). Stress factors induce alterations both alanopine, tauropine, octopine, and strombine (Ga in the amount of enzymes and in the production of isoforms possessing altered kinetic properties or post-translational modifications. In particular, since the estuarine and intertidal species are submitted naturally to multiple forms of daily stress, they display a wide range of metabolic adaptations, involving peculiar biochemical features (Storey and Storey, 1990;
* Corresponding author. Tel.: þ39 090 6765187; fax: þ39 090 6765186. E-mail address: [email protected] (D. Barreca). http://dx.doi.org/10.1016/j.bse.2014.05.020 0305-1978/© 2014 Elsevier Ltd. All rights reserved.
et al. / Biochemical Systematics and Ecology 56 (2014) 138e143 G. Lagana
139
lez-Wangüemerta et al., 2009). These adaptations of estuarine and Hochachka, 1997; Hochachka and Somero, 2002; Gonza intertidal bivalves to environmental variability have been poorly investigated in marine subtidal species, although they are €de and Grieshaber, 1986; frequently submitted to several types of stress causing short-term anaerobiosis (De Zwaan, 1977; Ga Fields and Storey, 1987). The Mediterranean endemic Pinna nobilis L., is one of the largest bivalves in the world. The “noble fan shell” lives partially buried in the soft bottom, with the broad posterior part of the shell that is elevated well above the sea floor. Fan shells are considered ecosystem engineers that remarkable increase habitat complexity, host epibiontic assemblages (Corriero and Pronzato, 1987; Cosentino and Giacobbe, 2008; Rabaoui et al., 2009) and locally influence oxygen and nutrient fluxes (Hewitt et al., 2006). Noble fan shell beds mostly occur from a maximum depth of 60 m in wave exposed areas up to 0.5 m in coastal lagoons (Zavodnik et al., 1991). In this respect, fan mussels might represent a profitable case-study to investigate metabolic adaptation of subtidal bivalves to environmental stress in oligotrophic shallow waters of sub-tropical areas (Butler et al., 1993). In this study, enzymatic markers and isozymes electrophoretic patterns of P. nobilis have been investigated in samples that are representative of a marine-brackish water gradient, to evaluate energetic metabolism and anaerobic adaptation in a long-lived moderately eurivalent species. 2. Methods 2.1. Sampling sites The specimens of P. nobilis were collected in the Straits of Messina area (central Mediterranean) between May and June of 2011, from two nearby marine and brackish-water sites; the respective populations, that are not spatially separated, in agreement with Butler et al. (1993), might belong to the same metapopulation. The marine sites included a wave exposed seafloor (St.1) and a more sheltered area (St.2), both at 7 m depth. The brackish water sites were located inside the channel that leads to the Faro lake (St.3) and in the inner part of the same lake (St.4), both at 1 m depth. In the Straits of Messina, the marine environment is characterized by tidal currents having a maximum speed of 3 m s1, with a period of 6 ¼ h. Such hydrological constraint leads to the upwelling of “Levantine Intermediate Water” that is colder, more salty and more nutrient-rich compared to the surface waters of Atlantic origin (Azzaro et al., 2007). This increase of primary production supports high density of benthic suspension feeders due to the water turbulences, oxygen availability and catabolytes clearing (Leonardi et al., 2009). The Faro lake is a meromictic basin, reaching a maximum depth of 29 m, characterized by anoxic and sulfidic waters, generally below 15 m in depth. Surface waters are mesotrophic, with a predominant heterotrophic biomass in the particulate matter and poorly oxygenated water can spread towards the surface in autumn. Exceptional seawater inflows cause strong alteration of the anoxic layer and temporary diseases on all aerobic organisms (Leonardi et al., 2009). 2.2. Sampling and sample treatment Adult specimens having a comparable shell size were collected manually in each site and transported alive, within 10 min, into the laboratory by means of a thermic bag. Once sacrificed, they were measured according to García-March and Ferrer (1995) and their biomass was evaluated as shell-free wet weight. Soft tissues were weighted, dissected, and frozen at 20 C until analysis. Adductor muscle, epatopancreas, gills and mantle were homogenized (1:4 w/v), using an Ultra Turrax tissue grinder, at 4 C in 0.01 M potassium phosphate buffer (pH 7) containing 0.5 mM phenylmethyl sulfonyl fluoride (PMSF) and 1 mM b-mercaptoethanol. The homogenate was centrifuged for 15 min at 12 000 rpm at 4 C. The supernatant was submitted to enzyme assays, protein determination and electrophoretic analysis. 2.3. Total protein and enzyme activities determinations Total protein content was determined according to Bradford (1976). The dehydrogenase activities were analyzed at et al. 25 ± 0.1 C following the changes of absorbance at 340 nm. LDH and MDH activity was assayed according to Lagana (2007). Alanopine (ADH), tauropine (TDH), strombine (SDH) and octopine (ODH) dehydrogenase activity was performed according to Baldwin et al. (1992). All the analyses were carried out in triplicate. One enzyme unit was defined as the amount catalyzing the production of 1.0 mM NAD þ per min at 25 C. In each experiment the values are the means ± standard deviation of three independent experiments (n ¼ 3). Statistical comparisons of the results were performed by one-way ANOVA. Significant differences (p < 0.05) between the tested samples were analyzed using Turkey’s test. 2.4. Isozymes determinations et al. (2007). LDH staining soVertical electrophoresis was performed on 7.5% polyacrylamide gel according to Lagana lutions consisted of 60 mM lithium lactate, 0.336 mM nicotinamide adenine dinucleotide (NADþ), 0.168 mM Nitro-blue tetrazolium (NBT), 0.056 mM phenazine methosulfate (PMS) in 0.1 M phosphate buffer (pH 7.0). MDH and ODH staining solution contained the same elements, but litium lactate was replaced with malate or octopine respectively. Staining of SOD was performed immediately after the achievement of electrophoresis using the nitrobluetetrazolium (NBT) photochemical method of Beauchamp and Fridovich (1971).
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Table 1 Protein (mg/g) content in the organs and tissues of the collected specimens. St.1 Epatopancreas Gills Adductor muscle Mantle
St.2
10.62 1.71 23.50 1.16
± ± ± ±
0.53a 0.38a 0.85a 0.3a
18.35 6.3 21.05 1.85
St.3 ± ± ± ±
1.26b 0.41b 1.39a 0.25a
15.50 6.00 27.08 3.75
St.4 ± ± ± ±
0.65c 0.24b 1.83b 0.46b
13.00 12.80 23.70 4.20
± ± ± ±
0.74d 0.95c 1.68a 0.72b
Different letters indicate significant difference (mean ± SD; n 3; p < 0.05).
3. Results The examined specimens ranged in size between 34 and 37 cm, with a biomass of 42 ge45 g. The marine specimens hosted the endo-commensalistic shrimp Pontonia pynnophylax, which was absent in the specimens collected in the lake and channel. The specimens collected in St.2, St.3 and St.4 showed hypertrophic gills and particularly those collected in the last station. Protein amount in the adductor muscle, epatopancreas, gills and mantle is summarized in Table 1. As can be seen epatopancreas and adductor muscles showed the highest protein content. Our survey of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) is summarized in Tables 2 and 3. It reveals marked differences both in the enzymatic activities and in the isoforms expression patterns. In all the analyzed tissues, MDH activity was usually much higher than the LDH one. The adductor muscle showed the highest level of MDH (4.73e6.19 U/g), followed by epatopancreas (0.86e4.43 U/g). Moreover samples collected in brachish habitat had higher levels of enzymes than those collected in the waves exposed station. Lactate dehydrogenase activity was found in dosable amounts only in the adductor muscle (0.33e0.65 U/g) and in the epatopancreas (0.74e0.98 U/g), while it was undetectable in the gills and in the mantle. In a different way from MDH, the samples collected in the waves exposed St.1 showed the highest level of LDH. The analyzed specimens had many opine dehydrogenases, whose function is similar to that of LDH. Fig. 1 shows the enzymatic activity of the tested opine dehydrogenases in the adductor muscle. These dehydrogenases can oxidize NADH produced by glycolysis and promote glycolysis to yield more ATP for physiological functioning under anoxia. The enzyme octopine dehydrogenase (ODH) showed the same tissue distribution evidenced by LDH, although the U/g were remarkably higher in the former instead of the latter. The adductor muscle also showed a conspicuous amount (U/g) of alanopine, tauropine and strombine dehydrogenase (Fig. 1). These enzymes were present only in trace in epatopancreas (data not shown). ODH is the major opine dehydrogenase in the adductor muscle, while ADH, SDH and TDH show almost the same activity. As can be seen from the graph, the U/g of ODH increased following the marine-brackish gradient (from specimens collected in St.1 to the ones collected in St.4). On the contrary, the U/g of the other analyzed opine dehydrogenases decreased following the same gradient (Fig. 1). The electrophoretic profiles of lactate, octopine and malate dehydrogenases are characterized by the presence of multiple isoenzymes. LDH isoenzymes are due to the A and B genes expression, resulting in the formation of homotetramers and one or more heterotetramers. The electrophoretic pattern generally showed the presence of three anodic bands only at the level of the adductor muscle and epatopancreas with little intensity differences between them (Fig. 2). In the epatopancreas of samples collected in St.4 no band was detected. The adductor muscle showed the presence of three bands in the specimens collected in St.1, St.2 and St.3, while only two bands were present in the samples collected in St.4 (Fig. 2). As in the case of LDH, octopine dehydrogenase pattern showed the presence of many bands only in the adductor muscle and in the epatopancreas. All the monitored isoforms were anodic, showing good eloctrophoretic mobility (Fig. 2). Only the samples collected in St.4 and St.1 showed significant bands at the level of the epatopancreas, consisting in two or three isozymes respectively (Rf between 0.63 and 0.74). Moreover the bands intensity was much more noticeable in the former than in the latter. In the adductor muscle there was the presence of at least three bands with good anodic electrophoretic mobility in all the samples. Moreover in the samples collected in St.4 there was a further isozyme, more anodic, with a Rf of 0.74. MDH activity is evident in all the samples analyzed, with clearly cathodic bands. Gills, epatopancreas and adductor muscle of the specimens collected in St.3 showed further isozymes with intermediate electrophoretic mobility, as well as the adductor muscle of the specimens collected in St.4. Seven isozymes were present in the gills of P. nobilis collected in St. 3, four similar to the ones present in the other samples and three characteristic of this habitat. A lower number of isozymes were present in the epatopancreas of the analyzed specimens with respect to the
Table 2 MDH activity (U/g) present in the organs and tissues of the collected specimens.
Epatopancreas Gills Adductor muscle Mantle
St.1 (wave exposed)
St.2 (sheltered)
St.3 (channel)
St.4 (lake)
MDH U/g
MDH U/g
MDH U/g
MDH U/g
0.86 0.35 4.73 0.52
± ± ± ±
0.03a 0.05a 0.12a 0.05a
3.21 1.40* 5.31 1.68
Different letters indicate significant difference (mean ± SD; n 3; p < 0.05). * hypertrophic; ** highly hypertrophic.
± ± ± ±
0.08b 0.10b 0.09b 0.07b
4.43 2.62* 6.19 1.09
± ± ± ±
0.12c 0.08c 0.09c 0.09c
1.58 1.65** 5.48 2.61
± ± ± ±
0.11d 0.07d 0.11b 0.1d
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Table 3 LDH activity (U/g) present in the organs and tissues of the collected specimens.
Epatopancreas Adductor muscle
St.1 (wave exposed)
St.2 (sheltered)
St.3 (channel)
St.4 (lake)
LDH U/g
LDH U/g
LDH U/g
LDH U/g
0.98 ± 0.06a 0.65 ± 0.07a
0.80 ± 0.08b 0.33 ± 0.05b
0.74 ± 0.04b 0.35 ± 0.05b
e 0.36 ± 0.07b
Different letters indicate significant difference (mean ± SD; n 3; p < 0.05). In gills and mantle we do not find detectable LDH activity.
gills. The mantle showed the presence of three main isozymes common to all the habitats, except for the samples collected in St.1 that had two bands corresponding to the more cathodic ones present in all the other samples. The electrophoretic pattern of the adductor muscle showed the presence of three cathodic isozymes (Rf of 0.09, 0.11 and 0.14). Moreover, the samples collected in St.3 and St.4 showed further less cathodic bands than the ones above mentioned, one coincident in the two habitats and another characteristic of the latter. The necessity to overcome oxidative injuries was highlighted by SOD electrophoretic pattern. Almost all the samples evidenced at least two SOD isozymes. In the gills were detected two bands common to all the habitats, except for those collected in St.4 that presented only one isoform. The specimens collected in St.2 and St.3 also showed a third band with higher mobility. Two SOD isozymes were present in the epatopancreas of the samples collected in St.1 and St.4, while those collected in St.2 and St.3 showed three bands. The mantle also showed the same electrophoretic pattern evidenced for the epatopancreas. The electrophoretic pattern of the adductor muscle showed two corresponding isozymes in the samples collected in St.1, St.2 and St.3; in this latter one was also present a third band with higher mobility. Only a cathodic band was present in the samples collected in St 4.
4. Discussion The marine intertidal and infaunal invertebrates give rise to biochemical changes and/or adaptive responses characteristic of the species and of the marine-brackish gradient. Assessment of biochemical features is essential to understand the influences of environmental changes on organism survival and to predict their interaction with the habitat. In all the tissues the expression of three separate gene loci (Mdh-M, Mdh-A and Mdh-B) of MDH was found. The presence of MDH-AB heterodimers, in almost all samples, indicates the simultaneous synthesis of both MDH-A and B subunits in many differentiated cells. Although the electrophoretic patterns exhibited essentially similar gene expression in the four tissues, they showed some habitat-specific isoforms. The most observed differences concerned the electrophoretic mobility and the relative intensity of the detected isozymes. In particular, isozyme electrophoretic patterns showed the presence of an anaerobic LDH isoenzyme and MDH isozymes (cytosolic and mitochondrial isoforms). The presence of opine dehydrogenases gives evidence of a remarkable metabolic plasticity and adaptability to environmental changes, according to literature data for estuarine and coastal bivalves (Hochachka and Somero, 2002). This is probably due to the energy need to overcome the changes associated with marine-brackish gradient. In fact opine dehydrogenase are able to produce more energy than the one that can be provided by the LDH route alone. The distribution of pyruvate reductases is strongly aligned with the ability to tolerate environmental hypoxia or to perform muscular work. Evidence from enzyme kinetics indicates that the ODH pathway is €de, 1983). Thus, ODH probably able to realize a higher rate of energy production than either the SDH or ADH pathways (Ga correlates with functional hypoxia during muscular activity, whereas SDH and LDH correlate with periods of environmental hypoxia. According to this data the ODH activity in adductor muscle increases following the marine-brackish gradient, while the one of ADH, SDH and TDH decreases following the same gradient. Moreover, anaerobic pathways are not all equivalent in terms of energy production based upon maximum rates for ATP output (lactate > octopine > alanopine ¼ strombine). For this reason, specimens utilizing the octopine (than alanopine) pathway will increase energy flow rapidly, developing a major ability to counteract environmental variations. This metabolic condition might conceivably facilitate a higher glycolytic rate and the production of a substrate (arginine) for the formation of octopine. In accordance with other mollusks that use a
Fig. 1. Opine dehydrogenases. Activities of ODH, ADH, SDH and TDH in the adductor muscle.
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Fig. 2. LDH and ODH isozymes. Electrophoretic pattern of LDH in epatopancreas and adductor muscle. The extracts were submitted to electrophoretic analysis and specifically staining for LDH or ODH activity.
variety of fermentation pathways, the absence of opine dehydrogenases in some tissues could be due to the utilization of the €rtner et al., 1984; Grieshaber et al., 1994). propionic acid pathway (Po The high ratio between MDH/LDH could be due to the ability of P. nobilis to turn on anaerobic metabolism as a consequence of environmental or anthropogenic stresses. High fluctuations of the thermo-aline regime, which especially affect the marineecontinental transition, explain the higher variability of enzymatic pattern, showed by the channel specimens. Recent studies have revealed that environmental temperature significantly affects both the total metabolic rate and metabolic € rtner, regulation in aquatic ectotherms, eliciting transition to anaerobiosis even in fully oxygenated waters (Sokolova and Po 2003). This experimental evidence is further supported by the electrophoretic patterns of SOD isozymes. Those showed the higher variability in the specimens collected in St.2 and St.3. In fact, it is well known that reactive oxygen species (ROS) are produced in cells subjected to environmental stresses such as exposure to intense light, UV radiations, metals, xenobiotics, thermal shock and change in salinity. Therefore, the induction of antioxidant enzymes like SOD is an important protective mechanism to minimize cell oxidation. In conclusion, the differences in the ability to withstand prolonged anoxia or rapid changes in environmental conditions (e.g. marine-brackish gradient) are correlated to a certain extent with the ecological peculiarities of the studied species and specifically with their biochemical pattern of enzyme expression. Acknowledgments The authors would like to thank William Fenton for his contribution in linguistic revision of the text. References Azzaro, F., Decembrini, F., Raffa, F., Crisafi, E., 2007. Seasonal variability of phytoplankton fluorescence in relation to the Straits of Messina (Sicily) tidal upwelling. Ocean. Sci. 3, 451e460. Baldwin, J., Wells, R.M.G., Low, M., Ryder, J.M., 1992. Tauropine and D-lactate as metabolic stress indicators during transport and storage of live Paua, (New Zealand Abalone) (Haliotis iris). J. Food Sci. 57, 280e282. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem 44, 276e287. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72, 248e254. Butler, A., Vicente, N., Gaulejac, B., 1993. Ecology of the pterioid bivalves Pinna bicolor Gmelin and Pinna nobilis L. Mar. Life 3, 37e45. Corriero, G., Pronzato, R., 1987. Epibiontic sponges on the bivalve Pinna nobilis. Mar. Ecol. Prog. Ser. 35, 75e82. Cosentino, A., Giacobbe, S., 2008. Aspects of epizoobiontic mollusc assemblages on Pinna shells. II. Does the Mediterranean P. nobilis represent an isle of biodiversity? Cah. Biol. Mar. 49, 161e173. De Zwaan, A., 1977. Anaerobic energy metabolism in bivalve molluscs. Oceanogr. Mar. Biol. A. Rev. 15, 103e187. Fields, J.H.A., Storey, K.B., 1987. Tissue-specific alanopine dehydrogenase from gill and strombine dehydrogenase from the foot muscle of the cherrystone clam Mercenaria rnercenana (Linn.). J. Exp. Mar. Biol. Ecol. 105, 175e185. €de, G., 1983. Energy metabolism of arthropods and mollusks during environmental and functional anaerobiosis. J. Exp. Zool. 228, 415e429. Ga €de, G., Grieshaber, M.K., 1986. Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates. Comp. Biochem. Physiol. 83, Ga 255e272. n dela equacio n de De Gaulejac y Vicente (1990). Bol. Inst. Esp. Oceanogr. 11, García-March, J.R., Ferrer, J., 1995. Biometría de Pinna nobilis L., 1758: una revisio 175e181. rez-Ruzafac, A., 2009. Phosphoglucose isomerase variability of Cerastoderma glaucum as a model Gonz alez-Wangüemerta, M., C anovasb, F., Marcosc, C., Pe for testing the influence of environmental conditions and dispersal patterns through quantitative ecology approaches. Biochem. Syst. Ecol. 37, 325e333.
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