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Mar Biol (2010) 157:718 DOI 10.

1007/s00227-009-1291-1

ORIGINAL PAPER

Effect of dissolved oxygen level on respiratory metabolism, nutritional physiology, and immune condition of southern king crab Lithodes santolla (Molina, 1782) (Decapoda, Lithodidae)
Kurt Paschke Juan Pablo Cumillaf Sergio Loyola Paulina Gebauer Mauricio Urbina Mara Eugenia Chimal Cristina Pascual Carlos Rosas

Received: 3 November 2008 / Accepted: 19 August 2009 / Published online: 3 September 2009 Springer-Verlag 2009

Abstract Episodes of hypoxia are common in the marine environment, and their ecological effects depend, in part, on their severity and duration. Many species of decapod crustaceans reside in areas with uctuating oxygen regimens. Physiological mechanisms enhance the ability of these crustaceans to cope with acute episodes of hypoxia. Southern king crab, Lithodes santolla, shery is important in the south of South America, and some data describe shing zones with low dissolved oxygen (DO) levels (3.5 mgO2 l-1, i.e., 8.3 kPa). Our main objective was to evaluate the effect of dissolved oxygen level on respiratory metabolism, nutritional physiology, and immunological condition of L. santolla juveniles. Individual animals were exposed for 10 days to different oxygen tensions (2.1, 4.2, 8.5, 12.7, and 21.1 kPa) to quantify the oxygen consumption rate; thereafter, blood oxyhemocyanin (Hc), protein concentration, as well as hemocytes, were sampled. Freezedried animals were dissected, and digestive gland metabolites (glycogen, protein, glucose, cholesterol, acylglycerol, and lactate) and digestive enzyme activity (general protease, trypsin, and chymotrypsin), as well as gill lactate dehydrogenase (LDH) activity, were quantied. In the
Communicated by H. O. Portner. K. Paschke (&) J. P. Cumillaf S. Loyola M. Urbina Instituto de Acuicultura, Universidad Austral de Chile, P.O. Box 1327, Puerto Montt, Chile e-mail: kpaschke@uach.cl P. Gebauer Centro de Investigacion I-Mar, Universidad de Los Lagos, Puerto Montt, Chile M. E. Chimal C. Pascual C. Rosas Unidad Multidisciplinaria de Docencia e Investigacion, UMDI, Puerto de Abrigo s/n, Sisal, Yucatan, Mexico

present study, Lithodes santolla showed a critical oxygen tension between 4 and 9 kPa, indicating that this crab species is more sensitive to DO than other crustacean species. Protein and Hc concentrations followed a similar pattern to that of oxygen consumption. Digestive gland glycogen and protein concentration did not change after 10 days at different oxygen exposures, but glucose, cholesterol, and acylglycerol concentrations decreased linearly and proportionally to the available oxygen in the water. As in other decapods, chymotrypsin showed over 90% of the total quantied proteases activity. Chymotrypsin activity together with total proteases and trypsin was not affected by the environmental oxygen tension. Gill LDH and digestive gland lactate followed a similar increase at lower environmental oxygen tension but dropped sharply at the lowest tension (2.1 kPa). Dissolved oxygen affected also the immune system through reduction of hemocytes. This could provide a critical window for opportunistic pathogens to become established when crabs are exposed to hypoxic conditions. L. santolla juveniles show a moderate tolerance to low oxygen availability by modifying the concentration of hemolymph proteins, mainly OxyHc, some digestive gland metabolites, and by activating the anaerobic metabolism. This allows L. santolla juveniles to inhabit temporarily low oxygen zones in the deep ocean and suggests an advantage for culture conditions.

Introduction Episodes of hypoxia are common in the marine environment, particularly associated to upwelling events (Grantham et al. 2004), as well as in shallow coastal zones. Coastal hypoxia generally follows seasonal patterns, some of them directly related to the inux of freshwater and

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anthropogenic eutrophication (Turner et al. 2008). Hypoxic zones are becoming more widespread and are one of the most deleterious human-induced impacts on benthic communities (Turner et al. 2008). Ecological effects of hypoxia depend, in part, on their severity and duration (Sagasti et al. 2001) and can disrupt benthic and demersal communities and cause mass mortality of aquatic life (Daz and Rosenberg 1995; Grantham et al. 2004). Generally, tolerance levels are higher for organisms residing in the sediments, whereas mobile organisms, such as sh and crustaceans, may exhibit behavioral responses to avoid hypoxic areas (Daz and Rosenberg 1995; Hagerman 1998). Moreover, oxygen supply does limit thermal toler ance in marine animals (Portner 2001, 2002, Portner et al. 2006). However, most organisms encountering hypoxic conditions have some physiological means of short-term adaptation. Many species of decapod crustaceans reside in areas with uctuating oxygen regimens. Physiological mechanisms enhance the ability of these crustaceans to cope with acute episodes of hypoxia. Many decapods are able to maintain oxygen uptake during hypoxia by increasing the ventilation of the branchial chambers (Airries and McMahon 1994; McMahon 2001). Below the critical oxygen tension (Pc), however, increases in ventilation rate are unable to compensate for hypoxia, and ventilation frequency decreases together with oxygen uptake (Airries and McMahon 1994). In hypoxic environments, most crustacean species also respond by exhibiting bradycardia, thus limiting the amount of energy expended by the cardiovascular system. Furthermore, crustaceans can alter blood ow during hypoxia, redirecting blood to tissues requiring higher levels of oxygen (McMahon and Wilkes 1975; De Souza and Taylor 1991; Airries and McMahon 1994; Reiber 1995; Reiber and McMahon 1998; McMahon 2001). In general, aquatic organisms are oxyregulators or oxygen conformers depending upon their ability to regulate metabolism as a function of oxygen concentration. For oxygen regulators, this ability and their behavior will be limited to the concentration of oxygen beneath which respiration follows the oxygen concentration (Vernberg 1983). This point has been dened as the incipient-limiting oxygen level (Fry 1947) and is referred as the critical oxygen level or Pc. Cherax destructor maintains hemolymph PO2 during progressive hypoxia by a fourfold hyperventilation (Morris and Callaghan 1998), whereas Litopenaeus setiferus reduces by 25% its oxygen consumption below 11.7 kPa (Rosas et al. 1997). The apparent critical partial pressure (Pc) of O2 in the water (PwO2) for ventilation and anaerobiosis in C. destructor (PwO2 \ 2.7 kPa) is comparable to that of other oxyregulating crustaceans (Morris and Callaghan 1998) but lower than for shrimp (Pc of 5.9 kPa; Rosas et al. 1998).

Recent studies made in other crustacean species demonstrated that oxygen consumption measurements are not sufcient to understand how an environmental factor, like dissolved oxygen (DO), modulates the physiological adaptation of benthic crustaceans. In fact, the hypoxiainducible factor (HIF), conserved during evolution from worms to ies to vertebrates, is central for adaptation to low oxygen availability (Semenza 1998). HIF regulates the transcription of many genes involved in the control of cellular and short- and long-term systemic responses to hypoxia, including glycolysis, erythropoiesis, breathing, vasodilatation, and angiogenesis in both vertebrates and invertebrates (Li and Brouwer 2007). Rosas et al. (1998) observed that a reduction in dissolved oxygen between 19.1 and 11.7 kPa produced a reduction in respiratory energy, but an increment in assimilated energy directed to production of biomass, showing that the effects produced by low dissolved oxygen levels are generally compensated by an increase in other physiological responses, despite reduced respiratory efciency. This type of response was also observed in Cancer magister, which tends to cease feeding below 3.2 kPa oxygen (McGaw and McMahon 2003). Adaptive responses to hypoxia include reduction in the metabolic rate (Hill 1976) and modications of the hemolymph acidbase balance (Martinez et al. 1998), hemocyanin binding capacity, oxyhemocyanin-protein relationship, hemolymph osmolality, and ion concentrations (Johnson and Uglow 1985; Charmantier et al. 1994; Chen and Kou 1998). Hemocyanin plays important roles in the binding and transport of oxygen and CO2, and as protein storage, carotenoids carrier, osmolyte, ecdysone transporter, and as a fungistatic. The synthesis of hemocyanin in crustaceans is enhanced by hypoxia in Carcinus maenas (Taylor and Anstiss 1999), Crangon crangon (Hagerman 1986), Nephrops norvegicus (Hagerman and Uglow 1985), Callinectes sapidus (DeFur and Mangum 1979), and Macrobrachium rosenbergii (Chen and Kou 1998). Hypoxia also induces hyperventilation, increasing water ow over gill surfaces for increased oxygen uptake and enhancing CO2 excretion from the hemolymph, which results in increased blood pH (Mauro and Malecha 1984; Johnson and Uglow 1987). In M. rosenbergii, hemolymph pH varies from 7.81 to 7.40 and 7.34 when oxygen partial pressure decreases from 80 to 40 and 15 Torr. At the same time, it was observed that lactate is accumulated in the hemolymph, suggesting that lactate together with CO2 accumulation could be responsible for the pH alterations (Mauro and Malecha 1984). A similar trend of hemolymph pH changes related with dissolved oxygen, lactate, and CO2 was also observed in Carcinus maenas (Johnson and Uglow 1987).

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Environmental variations induce changes in immune status of crustaceans, resulting in a reduction in immune vigor as measured by hemocyte counts, phagocytic indices, and release of free oxygen radicals (Le Moullac and Haffner 2000). In decapods, hemocytes are involved in phagocytosis that eliminates microbes or foreign particles (Bachere et al. 1995; Pascual et al. 2003b). Hemocytes are associated with proteins, like prophenoloxidase (proPO), which are involved in encapsulation, melanization, and cytotoxicity, as a nonself recognition system (Johansson et al. 2000). Environmental parameters like high pH and low dissolved oxygen (DO) have been reported to cause reduction in hemocyte counts in Macrobrachium rosenbergii (Cheng and Chen 2000) and in the blue shrimp Litopenaeus stylirostris (Le Moullac et al. 1998). Recent studies have demonstrated that fed animals had different responses to low dissolved oxygen levels than animals maintained in fasting conditions. Results obtained in Cancer magister demonstrated that the nutritional state of crabs is important in modulating their physiological responses to low DO. When animals were fed and exposed to hypoxia a reduction in bradycardia was observed, while an increment in ventilation rate was recorded. At the same time, a reduction in cardiac output was noted together with a blood ow diversion away from the hepatopancreas. A reduction in protein synthesis was interpreted as a consequence of that response (McGaw 2005). Taking into account that macrofauna and megafauna often exhibit dense aggregations at oxygen minimum zone edges due to the organic rich sediments of these regions (Levin 2003), the study of physiological responses of benthic organisms acclimated and fed under hypoxic conditions are important to known how adverse environments modulate crustacean physiology. Lithodes santolla (southern king crab) is one of the most important shellsh sheries in Tierra del Fuego, Argentina, and in the XII Region, Chile. Although no information is available on many biological aspects related to its larval distribution and information on recruitment zones is scarce (Tapella and Lovrich 2006), there is evidence indicating that the dissolved oxygen could be lower than 8.3 kPa in the zones where adults live (Guzman and Silva 2002; Silva and Guzman 2006). In the present study, the effect of dissolved oxygen (DO) on different physiological responses of L. santolla was evaluated in an attempt to know how the Pc and DO modulate several other aspects of nutrition (blood proteins, digestive gland metabolites, and oxyhemocyanin: OxyHc), immune state (hemocytes concentration), digestive capacity (general protease, trypsin, and chymotrypsin), and metabolic enzymes (lactate dehydrogenase: LDH) of crabs exposed to long term DO levels of 21.1, 12.7, 8.5, 4.2, and 2.1 kPa.

Materials and methods Animals Lithodes santolla juveniles (3.3 0.11 g wet weight; ca. 1-year-old animals) were obtained from natural spawns, and larvae were cultivated at the Universidad Austral de Chile, Puerto Montt, Chile. Larvae and juveniles were cultivated in sea water at 12 0.5C, 31 psu, and an oxygen concentration higher than 8 mgO2 l-1 ([21 kPa; oxygen equivalencies following Colt 1984). Experimental design For 10 days, 10 juveniles were reared in 34-l hermetic chambers at 2.1, 4.2, 8.5, 12.7, and 21.1 kPa (rounded as 2, 4, 9, 13, and 21 kPa) (a total of 50 individuals), at 12C and 31 psu. Twice a day, 1-lm UV ltered sea water was exchanged at the corresponding oxygen concentration. Nitrogen was used to reach the sea waters DO at each DO experimental level. To maintain DO levels constant, the sea water in the sealed chambers was exchanged twice a day, at 0800 and 1600 hour. Crabs were fed once a day (0900 hour) with Chilean mussel (Mytilus chilensis) pieces, and nonconsumed food and feces were removed after 6 h. Survival was recorded daily. After 10 days of DO experimental level exposure, oxygen consumption, blood metabolites, hemocytes concentration, digestive gland biochemical characterization, and gill LDH activity were evaluated. Oxygen consumption Seven to ten animals per treatment were incubated individually in sea water (0.22-lm ltered UV-treated seawater at 12C and 31 psu) in 1,000-ml hermetic chambers at each experimental DO level (closed respirometer). Oxygen content was quantied before and after incubation (ca. 3 h) by an optic sensor connected to a temperature-compensated Microx MX3 AOT oxygen meter (PreSens, GmbH, Germany) calibrated with saturated sea water (100%) and 5% sodium sulte solution (0%). Three chambers without animals were used per treatment as control. Blood analysis Hemolymph sampling After oxygen consumption measurements, ve crabs were randomly sampled for OxyHc, hemocytes concentration, and blood proteins. Thereafter, each animal was frozen at -30C and freeze dried for 48 h. Hemolymph was sampled individually with a chilled syringe needle inserted at the

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abdominal sinus, after drying the crab with a paper towel. To avoid clotting, blood samples were taken without air because air accelerates the coagulation process. Blood samples of each crab were placed gently on chilled (4C) plastic foil (Paralm@) over ice. Subsamples were taken from the hemolymph samples with a micropipette for OxyHc, blood protein, and hemocytes concentration. Hemocyanin and protein level For OxyHc measurements, 20 ll of hemolymph was diluted immediately with 980 ll of distilled water in a 10-mm cuvette (1.0 ml; 1-cm path length), and the absorbance was measured at 335 nm. Using an extinction coefcient of e = 17.26, calculated on the basis of the functional subunit of 74,000 Da for crab, hemocyanin concentration was determined (Chen and Cheng 1993a, b). Total protein was measured in 500 ll hemolymph diluted immediately with 1 ml of precooled (4C) anticoagulant (450 mM NaCl, 10 mM KCl, 10 mM HEPES, 10 mM EDTA-Na2, pH 7.3, 850 mOsm kg-1) (VargasAlbores et al. 1993). Hemolymph plus anticoagulant was centrifuged at 800g for 3 min at 4C, and the supernatant was separated for protein determinations. Blood protein concentration was obtained using a desk refraction meter (Atago), previously calibrated using bovine serum albumin as standard solution. Hemocytes level

discarded, leaving the glycogen as a pellet; glycogen was dissolved by adding 1 ml concentrated sulfuric acid and 200 ll phenol (5%). From this mixture, three samples of 200 ll were transferred to a microplate and read at 490 nm in an ELISA reader (Biorad 550). Total weight of the digestive gland was also recorded. Glycogen was obtained as glucose in the sample using a glucose standard from a commercial kit (GOD-PAD, Merck-740393). Glycogen was expressed as milligrams of glycogen per gram of tissue. A second subsample of the digestive gland was rehydrated using pyrogen-free water (2:8; w:v) and homogenized in an assay tube placed in an ice bath. Assay tubes were centrifuged for 10 min at 8,000g and 4C. Commercial kits were used for glucose (GOD-PAD, Merck-740393), lactate (Sigma-cat. 735), cholesterol (CHOD-PAP, Merck, cat. 14349), and acylglycerol (GPO-PAP, Merck, cat. 14354). The kits method was adapted to a microplate using 20 ll of digestive gland homogenates and 200 ll of enzyme chromogen reagent. Each sample was assayed in triplicate for metabolite assessment. Absorbance was recorded on a microplate reader (BIO-RAD model 550), and concentrations were calculated from a standard solution of substrate. Digestive gland homogenates were further diluted 1:500 for protein determination by the Bradford technique (Bradford 1976) adapted to a microplate method using a commercial chromogen reagent (Sigma, cat. 610) and bovine serum albumin as standard solution. Digestive enzyme activity

Hemolymph samples (150 ll) were mixed with 450 ll of Alsever solution (113 mM glucose, 27.2 mM sodium citrate, 2.8 mM citric acid, 71.9 mM NaCl) supplemented with 10% formaldehyde (v/v). These samples were stored at 4C until analysis. Total hemocyte counts were done in a Neubauer chamber. Tissues analysis Biochemical characterization of the digestive gland metabolites Glycogen in the digestive gland (Gly) was extracted with trichloroacetic acid (TCA) and determined through the reaction with sulfuric acid and phenol (Dubois et al. 1956). The digestive gland was dissected, and a section was weighed (21 mg) and homogenized in trichloroacetic acid (5% TCA) for 6 min at 8,000g (Micro Centrifuge Eppendorf 5415). From the supernatant, 100 ll was pipetted into an assay tube and mixed with ve volumes of 95% ethanol. Assay tubes were placed in an oven at 3740C for 3 h. After precipitation, the assay tubes were centrifuged at 3,340g for 15 min. The supernatant was

General proteases activity was estimated in crude digestive gland homogenates using azocoll (Sigma A4341) as substrate in phosphate buffer, pH 7.5. Absorbance was measured in a spectrophotometer (SPECTRONIC model 21D) at 520 nm. For this method, one unit was dened as the amount of enzyme that catalyses the release of azo dye causing a DA/Dt = 0.001 DO min-1 (Walter 1988). Each sample was assayed in duplicate. Trypsin-like enzyme activity was assayed in crude homogenates using 100 mM Na-benzoyl-DL-arginine p-nitroanilide (BAPNA, Sigma B4875) as substrate in 0.1 M Tris buffer, pH 8. The change in absorbance was measured for 2 min at 405 nm. Chymotrypsin enzyme activity was assayed in crude homogenates with N-succinyl-ala-ala-pro-phe p-nitroanilide (SAAPPNA, Sigma S7388) as substrate in 0.1 M TRIS buffer, pH 8. The change in absorbance was measured over 2 min at 405 nm. One unit of trypsin and chymotrypsin activity corresponded to 1 lM of 4-nitroaniline liberated in 1 min, based on an extinction coefcient of e405 = 1.02 l mmol-1 mm-1 (Geiger and Fritz 1988). Enzymatic activity was expressed as international units (IU) per milligram of protein.

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Gills (8 2.1 mg) were rehydrated using 200 ll pyrogenfree water (2:8; w:v) and homogenized in an assay tube placed in an ice bath. LDH activity was measured in crude homogenates using a commercial kit (lactate dehydrogenase, Diagnostic Chemicals Limited, Chalottetown, PE, Canada) based on the proportional increase in absorbance at 340 nm due to the formation of NADH. Samples were centrifuged at 8,000g and 4C for 20 min, and 10 ll of supernatant was added to 1 ml LDH reagent. LDH reagent was preincubated at 37C for 5 min, after the addition of 6.5 ml pyrogen-free water. Each sample was assayed in triplicate. The change in absorbance was measured each minute for 8 min at 340 nm. Enzymatic activity was expressed as international units per milligram of protein, considering an absorption coefcient of 6.22 for NADH. Gill proteins were quantied similarly to digestive gland proteins. Statistical analysis Normal distribution and homogeneity of variances were tested with KolmogorovSmirnov and Levene median tests, respectively. When the data did not satisfy the prerequisites for parametric tests (analysis of variance, ANOVA), KruskalWallis H-tests were used to evaluate the effect of oxygen tension on oxygen consumption, oxyhemocyanin concentration, hemolymph protein concentration, hemocytes level, digestive gland metabolites, and enzymatic activity. When the probability of error for rejecting the null hypothesis was higher than 0.05, differences were considered not signicant (NS). Otherwise, a posteriori HolmSidak or Dunn test, for parametric and nonparametric analyses, respectively, was conducted to identify different treatments. Digestive gland metabolites, such as glucose, cholesterol, and acylglycerol, were tted to a linear regression with respect to the oxygen tension, using the least-squares method. Other metabolites did not satisfy the prerequisites for linear tting. An R*C test of independence was used to analyze effects of treatments on mortality (Sokal and Rohlf 1995).

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Fig. 1 L. santolla. Individual oxygen consumption rate related to dissolved oxygen level. Mean standard deviation. Signicant differences among treatments indicated by different letters (ANOVA, P \ 0.05)

Oxygen consumption Oxygen consumption was constant in animals exposed to 9, 13, and 21 kPa with a mean value of 0.41 0.16 mgO2 h-1 ind-1(P [ 0.05; Fig. 1). A reduction in oxygen consumption was observed with DO lower than 9 kPa, yielding values of 0.24 0.07 mgO2 h-1 ind-1 (4 kPa DO) and 0.13 0.02 mgO2 h-1 ind-1 (2 kPa DO) (P \ 0.05). On the basis of these results, a critical oxygen tension (Pc) for juveniles of Lithodes santolla of around 9 kPa can be proposed (Fig. 1). Blood analysis Hemocyanin and protein level No statistical differences in OxyHc values were found in crabs exposed to 9, 13, and 21 kPa (18.17 3.28 mg ml-1), resulting 100% higher than the values observed in animals maintained at 2 and 4 kPa (6.86 5.5 and 11.13 3.77 mg ml-1, respectively; mean value of 8.99 3.0 mg ml-1; P \ 0.05; Fig. 2). Although a nonsignicant increase was observed, at 13 kPa the highest OxyHc values were recorded, reaching 21.6 11.5 mg ml-1, i.e., 43% of the 21 kPa treatment. A mean value of 51.3 9.84 mg ml-1 of protein was observed in animals maintained at 9, 13, and 21 kPa (P [ 0.05; Fig. 3). At DO levels lower than 9 kPa, a reduction in blood protein level was registered with values of 37.5 mg ml-1 (at 4 kPa) and of 30.3 mg ml-1 (at 2 kPa); (P \ 0.05; Fig. 2). Hemocytes level Total hemocytes were not affected by DO levels in animals maintained at 4, 9, 13, and 21 kPa (mean value of 1,549 cell mm-3; P [ 0.05). In contrast, a low value of

Results No signicant differences were detected in size and weight of crabs from the ve experimental treatments (ANOVA, P [ 0.05). After 10 days of different experimental DO levels, almost all crabs survived even at an oxygen tension of 2 kPa (R*C gH test, P [ 0.05; 100% survival with treatments 4 and 21 kPa; 90% survival with treatments 9 and 13 kPa; 70% survival with treatment 2 kPa).

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Fig. 2 L. santolla. Hemolymph protein (Prot) and oxyhemocyanin (Oxy-Hc) concentrations related to dissolved oxygen level. Mean standard deviation. Signicant differences between treatments indicated by different letters (ANOVA, P \ 0.05)
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Fig. 3 L. santolla. Hemocytes concentration related to dissolved oxygen level. Mean standard deviation. Signicant differences between treatments indicated by different letters (ANOVA, P \ 0.05)

Fig. 4 L. santolla. Digestive enzymes activity related to dissolved oxygen level. a Total proteases, b trypsin activity, and c chymotrypsin activity. Mean standard deviation. No signicant differences among treatments for the three variables (ANOVA, P [ 0.05)

hemocytes (611 cell mm-3) was registered in animals exposed to 2 kPa, resulting 60% lower than that observed in animals exposed to the rest of the DO treatments (P \ 0.05; Fig. 3). Biochemical characterization of the digestive gland Digestive enzyme activity Digestive (proteolytic) enzymes were not affected by low DO levels (P [ 0.05; Fig. 4a). Trypsin activity uctuated around 388 135 IU mg protein-1 (Fig. 4b), whereas chymotrypsin showed a mean value of 14,437 5,356 IU mg protein-1 (Fig. 4c). Metabolites Although a 47% decrease in digestive gland glycogen (30.4 18.4 mg gW-1) and decrease of 7.6% in total

soluble protein (77.0 7.8 mg gW-1) were observed at the lowest DO, no signicant differences were established (P [ 0.05; Fig. 5a; mean values at DO levels between 2 and 21 kPa: glycogen 49.4 18.9 mg gW-1, protein 85.35 16.01 mg gW-1). In contrast, glucose, acylglycerol, and cholesterol (mg gW-1) showed a direct linear relationship with DO values, being low in animals exposed to low DO levels and high in animals exposed to 21 kPa (P \ 0.05; Fig. 5b). The relationship between DO and these blood metabolites is described by the following equations: Glucose (mg gW1 5:836 0:352 DO; r 2 0:343; P 0:005 Cholesterol (mg gW1 2:657 0:233 DO; r 2 0:262; P 0:012 Acylglycerol (mg gW1 15:947 2:335 DO; r 2 0:221; P 0:021

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Fig. 5 L. santolla. Metabolites in digestive gland related to dissolved oxygen level. a Protein (Prot) and glycogen (Glyc) content (ANOVA, P [ 0.05). Mean standard deviation. b Glucose (Gluc), cholesterol (Chol), left axis, and acylglycerols (AG), right axis, contents. Mean ? standard deviation. Linear regression coefcients see text (P \ 0.05)

Lactate in digestive gland and gill LDH Digestive gland lactate increased when DO declined, reaching its highest value in animals exposed to 4 kPa (2.38 0.94 mg gW-1), although, at 2 kPa DO level, lactate concentration dropped to 1.82 0.69 mg gW-1 (Fig. 6). A similar behavior was observed in gill LDH. LDH activity increased with decreasing DO levels, being low in animals maintained at 21 kPa (13.6 1.2 UI mg protein-1) and high in animals maintained at 4 kPa (83.7 65.7 UI mg protein-1) (P \ 0.05; Fig. 6). However, a reduction in LDH activity was registered in animals exposed to 2 kPa (P \ 0.05).

Discussion In the present study, a Pc between 4 and 9 kPa was obtained for L. santolla, indicating that this crab species is more sensitive to DO than other crustacean species. The apparent critical partial pressure (Pc) of O2 in the water

(PwO2) for ventilation and anaerobiosis in Cherax destructor is lower than 2.7 kPa (Morris and Callaghan 1998), and it is around 5.9 kPa for L. setiferus shrimp (Rosas et al. 1998). An analysis of the Pc of a wide variety of pelagic crustaceans living at minimum oxygen layer depths in different oceanic habits showed that there are two groups: organisms able to maintain their aerobic metabolic rates even at the lowest DO concentration in their environments and organisms whose Pc is higher than or similar to the lowest DO concentration in their environments (Childress and Seibel 1998). These authors suggested that, for the rst group of crustaceans, values below 4 kPa could indicate specic adaptations related with their ability to maintain their aerobic metabolism at low DO. Values higher than 4 kPa seem to be typical of animals living at high environmental oxygen levels that, in consequence, could have limited adaptation capability to respond to lower DO levels. In the present study, L. santolla showed a Pc value between 4 and 9 kPa, indicating that this species could be classied into the second group of crustaceans because such a Pc interval could be around or higher than the minimum DO concentration reported for the zone of adult crab distribution (3.5 mgO2 l-1: 8.3 kPa; Guzman and Silva 2002; Silva and Guzman 2006). Grieshaber et al. (1994) suggested that the transition to oxyconformity in oxyregulators is mainly linked to the onset of anaerobic processes and Childress and Seibel (1998) suggest that adaptations to low oxygen partial pressures include anaerobic metabolism and mechanisms to facilitate oxygen uptake. With these mechanisms, crustaceans respond to hypoxia by regulating oxygen transport and increasing cardiac output and hemocyanin synthesis (Mangum 1997; Paul et al. 2004). Some of these responses have been observed in several crustacean species, both fresh water and marine species (Morris and Callaghan 1998; Bridges 2001; Brown-Peterson et al. 2005).

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In the present study, a change in OxyHc concentration was observed according to DO level changes, with slightly higher values in animals maintained between 9 and 21 kPa in comparison with treatments with lower DO. In this situation, L. santolla was able to turn on mechanism to either enhance the afnity of the actual OxyHc concentration or increase the hemocyanin level. In the rst case, the OxyHc afnity can be obtained via the increment of lactate that counteracts the Bohr effect in hypoxic conditions (Bridges 2001). Hc of early juveniles of Cancer magister showed 50% lower afnity than adult Hc, and it is very sensitive to magnesium concentration (Terwilliger and Dumler 2001). Energy restriction provoked by low DO alters homeostasis in L. santolla juveniles (such as hemolymphatic constituents) and eventually Mg concentration, affecting Hc afnity. Another option is that hemocyanin synthesis acts as a primary response to prevent cellular hypoxia. Although, in the present study, a signicant increment on OxyHc was not observed in animals maintained between 21 and 9 kPa, a high value was recorded in animals maintained at 13 kPa, suggesting that at that oxygen level L. santolla can synthesize OxyHc as a rst line response when DO is reduced to this range. In DO levels lower than 9 kPa, the OxyHc concentration was reduced following a reduction in total hemolymphatic protein, suggesting that the mechanisms involved in Hc synthesis will be markedly affected at DO levels lower than 9 kPa. A recent study (Brouwer et al. 2007) showed that, after a 3-days exposure to severe hypoxia, Palaemonetes pugio was able to upregulate genes of ATP synthesis-d- and ATP synthesis-fchains (ATPsyn-d and ATPsyn-f), three hemocyanin genes (Hcy II, Hcy III, and Hcy IV), troponin C, and ferritin. That study suggested that an attempt of the shrimp to increase oxygen uptake/transport (hemocyanin), ATP synthesis, and locomotion (troponin C) could be exerted as a rst response to low DO levels. These authors observed also that after a 7-day exposure to chronic hypoxia, the adaptation induced by the 3-day exposure becomes insufcient, and ATP synthetase, hemocyanin, and troponin are no longer up-regulated. These results could be used to explain how hemocyanin of L. santolla decreased when DO dropped below 9 kPa. This apparent contradiction in reducing OxyHc at lower DO when more is needed may result from a diminished energy supply. Hc concentration is very dynamic, modulated by stressors, temperature, nutritional status, DO, and seems to be undergoing an almost continuous synthesis. Under diminished energy input provoked by reduced food ingestion at low DO, the rate of synthesis of Hc could be affected, even more than other hemolymphatic proteins. As in P. pugio (Brouwer et al. 2007), OxyHc levels registered at DO tension below 9 kPa suggest that transcription might be turned off, i.e.,

gene regulation, and that other mechanisms were involved in OxyHc regulation. Hemocyanin is not only regulated by dissolved oxygen. In shrimp, OxyHc concentration depends on nutritional characteristics of the diet, indicating that its synthesis is modulated mainly by dietary protein and the nutritional condition (Senkbeil and Wriston 1981; Chen and Cheng 1993a, b; Chen et al. 1994; Pascual et al. 2003a). At the same time, many reports indicate that DO affects food consumption of crustaceans mainly due to a reduction in the energy directed to the digestive process. A reduction in the energy invested in the ingestion rate and specic dynamic action (SDA, i.e., increase in oxygen consumption related to digestion processes) was observed in Litopenaeus setiferus maintained at DO concentrations below Pc levels (Rosas et al. 1998). This reduction in the ingestion rate observed at low DO concentrations could be related with how blood is diverted away from digestive structures during feeding, affecting the nutritional condition of crabs (McGaw 2005) and the synthesis of Hc, as stated for Astacus leptodactylus (Gellisen et al. 1991). Although, in the present study, blood ow during hypoxia was not measured, reduction in digestive gland metabolites (glucose, acylglycerol, and cholesterol), blood protein, and OxyHc under hypoxia demonstrates that the reduction in the ingestion rate, due to the reduced dissolved oxygen, affected the nutritional condition of crabs and protein synthesis. Ingested rate of Lithodes santolla juveniles was estimated, on the basis of dry weight, by daily differences in recovered and offered Mytilus chilensis pieces. Mean values standard deviation for the 10-day incubation period revealed two major groups: 24 kPa (0.597 0.709, 0.949 1.019 mg W h-1 ind-1, respectively) and 921 kPa (2.798 1.985, 3.737 1.916, 3.901 1.974 mg W h-1 ind-1 for 9, 13 and 21 kPa, respectively) (ANOVA P \ 0.05, HolmSidak method). A similar reduction in digestive gland metabolites, as observed in L. santolla maintained at 2 kPa, was found in shrimp maintained under starvation conditions for 7 14 days, demonstrating that lack of food and dissolved oxygen regulate the nutritional condition of crustaceans (Pascual et al. 2006). In this sense, Bernatis et al. (2007) showed that C. magister preferred an oxygen tension range between 8 and 17 kPa, but they would enter into and feed in severe hypoxic waters. Although C. magister is able to use physiological mechanisms to control digestive processes in hypoxia (McGaw 2005), it is evident from the current results that the physiological mechanisms involved in the response under hypoxia in fed L. santolla affect the synthesis of molecules, such as protein or hemocyanin, with important roles in the physiological adaptations of crabs.

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The activity of the digestive enzymes was not affected by the oxygen tension and, as reported (Navarrete del Toro et al. 2006; Saborowski et al. 2006), chymotrypsin resulted the most important protease, when compared with the activity of trypsin. In the present study, low DO concentration induced a reduction in digestive processes because crabs diminished food ingestion. A probable mechanistic link could be the muscular work for food uptake, which has to be fueled by ATP synthesis that is limited by oxygen supply and energy input, rather than the capability of enzymatic digestion. Activity of L. santolla digestive enzymes remained constant, suggesting a hypothesis to explain such results: crabs are able to react to the food supply immediately due to the unaltered activity of proteolytic enzymes. For shrimp exposed during short-term starvation (120 h), Sanchez-Paz et al. (2007) showed that plasmatic proteins remain constant during the experiment and concluded that protein mobilization between muscle reserves and digestive gland could be used as a physiological strategy to maintain shrimp during short fasting periods. In another study, Muhlia-Almazan and Garca Carreno (2002) showed that total digestive proteases, trypsin, and chymotrypsin of L. vannamei remained constant during 120-h starvation, indicating that activity, as well as synthesis of digestive proteases, is maintained constant. A similar strategy could be operating in L. santolla during the reduced ingestion period provoked by low DO levels. Total blood protein concentration of L. santolla did not change from 9 to 21 kPa, but a reduction in blood protein was observed in animals exposed to 4 and 2 kPa, suggesting a blood protein Pc of 9 kPa. Although blood protein is mainly constituted by hemocyanin (6090%) (Pascual et al. 2003a; Rosas et al. 2004), values lower than 50% have been reported for Scylla serrata (Chen and Chia 1997) and Penaeus monodon (Chen and Cheng 1993a), suggesting that OxyHc/protein proportion depends on the type of organisms, nutritional condition, and ecological condition, among others. In the present study, OxyHc/protein proportion in L. santolla control group showed values (30.6%; 21 kPa DO) lower than those reported for shrimp, suggesting species-specic physiological adaptations related with the ecology of this crab species. Preliminary observations made in our laboratory indicate that L. santolla could have a high volume of circulating hemolymph, which could maintain all blood components in a high degree of dilution. Although to test this hypothesis, hemolymph content of L. santolla should be determined. OxyHc concentrations similar to that observed in the present study (0.2 mmol l-1) were also observed in other crustacean species from cold and deep waters, such as Homarus gammarus (Hagerman and Uglow 1985), H. arenaeus (0.2 mmol l-1), Nephrops norvegicus (0.39 mmol l-1), and

Liocarcinus depurator (Spicer and Baden 2000). This similitude suggests that the relatively low OxyHc/protein proportion could be a common condition among crustacean species. OxyHc/protein proportions were affected by DO and followed the same behavior as that observed for OxyHc values: low values in animals maintained at 2 kPa and high values in animals maintained at 13 kPa, supporting the idea that both OxyHc and protein were modulated by the way in which blood ow is diverted away from the digestive gland, the main site for hemocyanin and protein synthesis (Engel and Brouwer 1991). Besides hemocyanin, there are other important proteins that circulate in the blood stream. Blood proteins are involved in the immune system by recognizing foreign glucans through the lipopolysaccharide-binding protein (LPSBP), the b-glucan-binding protein, and other lectins (Yepiz-Plascencia et al. 2000; Alpuche et al. 2005). In addition, there are enzymes involved in melanin formation (phenoloxidase) and its regulators (trypsin, alpha-2-macroglobulin, and pacifastin), as well as peptides and soluble proteins from nutritional origin (Capuzzo and Lancaster 1979; Glass and Stark 1994; Chuang et al. 1995). Taking into account that hormones, nutritional peptides, and immune components are part of the total blood proteins, their quantication has been used as an indicator of the health status of several crustacean species (Sanchez 2001; Pascual et al. 2003b, 2004a, b, 2006; Rosas et al. 2004). The oxygen tension of 9 kPa as a Pc for blood protein and for oxygen consumption suggests that, like other crustaceans, the homeostasis of L. santolla is a protein-dependent mechanism that is able to maintain stable its metabolism under a DO range higher than the minimum dissolved oxygen reported for its distribution area. A relationship between digestive gland protein and glycogen was studied in order to explore if in L. santolla, as in other crustacean species, glycogen follows the gluconeogenesis pathway (Meenaski and Sheer 1961; Wang and Scheer 1963; McWhinnie and Corkill 1964; Rosas et al. 2002). A linear inverse relationship was found between digestive gland glycogen and protein [glycogen = 87.369(0.414 protein); r2 = 0.161, P = 0.045], suggesting that glycogen synthesis was a result of digestive gland protein degradation during glycogen synthesis. Although in the present study it is not clear how this type of biochemical pathway was modulated, it is possible to think that it resulted from the nutritional condition and DO effects that, at the same time, modulated the use of energy by L. santolla. The number of circulating hemocytes in L. santolla was affected by hypoxia, showing low-cell concentration in animals maintained at 2 kPa. According to Pascual et al. (2004a, b) and (2006), hemocytes can be regulated by the nutritional condition, including dietary protein level,

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Mar Biol (2010) 157:718 Bernatis JL, Gerstenberger S, McGaw IJ (2007) Behavioural responses of Dungeness crabs, Cancer magister, during feeding and digestion in hypoxia. Mar Biol 150:941951 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254 Bridges CR (2001) Modulation of haemocyanin oxygen afnity: properties and physiological implications in a changing world. J Exp Biol 204:10211032 Brouwer M, Brown-Peterson NJ, Larkin P, Patel V, Denslow N, Manning S, Brouwer TH (2007) Molecular and whole animal responses of grass shrimp, Palaemonetes pugio, exposed to chronic hypoxia. J Exp Mar Biol Ecol 341:1631 Brown-Peterson NJ, Larkin P, Denslow N, King C, Manning S, Brouwer M (2005) Molecular indicators of hypoxia in blue crab, Callinectes sapidus. Mar Ecol Prog Ser 288:203215 Capuzzo JM, Lancaster BA (1979) The effects of diet on the growth energetics of postlarval lobsters Homarus americanus. Proc. World Maric. Soc. 10:689700 Charmantier G, Soyez C, Aquacop (1994) Effect of moult stage and hypoxia on osmoregulatory capacity in the peneid shrimp Penaeus vannamei. J Exp Mar Biol Ecol 178:223246 Chen JC, Cheng S-Y (1993a) Hemolymph PCO2, hemocyanin, protein level and urea excretions of Penaeus monodon exposed to ambient ammonia. Aquat Toxicol 27:281292 Chen JC, Cheng S-Y (1993b) Studies in hemocyanin and hemolymph protein levels of Penaeus japonicus based on sex, size and moulting cycle. Comp Biochem Physiol 106B:293296 Chen JC, Chia PG (1997) Oxyhemocyanin, protein, osmolality and electrolyte levels in the hemolymph of Scylla serrata in relation to size and molt cycle. J Exp Mar Biol Ecol 217:93105 Chen JC, Kou TT (1998) Hemolymph acidbase balance, oxyhemocyanin, and protein levels of Macrobrachium rosenbergii at different concentrations of dissolved oxygen. J Crustacean Biol 18:437441 Chen JC, Cheng S-Y, Chen CT (1994) Changes of hemocyanin, protein and free aminoacid levels in the hemolymph of Penaeus japonicus exposed to ambient ammonia. Comp Biochem Physiol 109A:339347 Cheng W, Chen JC (2000) Effects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish Shellsh Immunol 10:387391 Childress JJ, Seibel BA (1998) Life at stable low oxygen levels: adaptations of animals to oceanic oxygen minimum layers. J Exp Biol 201:12231232 Chuang NN, Lin CL, Chen HK (1995) Modication of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda). Comp Biochem Physiol B 114B(2):145151 Colt J (1984) Computation of dissolved gas concentrations in water as functions of temperature, salinity, and pressure. American Fisheries Society Special Publication 14 De Souza SCR, Taylor EW (1991) Electrolytes and acidbase regulation in Carcinus maenas during prolonged aerial exposure. J Physiol 435:106P DeFur PL, Mangum CP (1979) The effects of environmental variables on the heart rates of invertebrates. Comp Biochem Physiol 62A:283294 Daz RJ, Rosenberg R (1995) Marine benthic hypoxia. A review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanogr Mar Biol Annu Rev 33:245303 Dubois MK, Lilles LA, Hamilton JC, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Analyt Chem 28:350356

carbohydrate level, and fasting. During the experiment, L. santolla exposed to 2 kPa practically did not ingest food, indicating that, at this DO level, crabs were nutritionally affected and, in consequence, the synthesis of circulating hemocytes. A study with Cancer magister demonstrated that, during hypoxia, hemolymph ow rates through the supra-esophageal ganglion did not change during feeding, suggesting that the supra-esophageal ganglion was prioritized independently from the DO level (McGaw 2005). Such a prioritization could affect hemocytes synthesis. Studies made in shrimps demonstrated that the hematopoietic tissue is located close to the supra-esophageal ganglion and covers the dorsal and dorsolateral sides of the stomach, surrounded by connective tissue (Johansson et al. 2000). Current results evidence that the prioritization of blood ow during hypoxia could be directed to preserve the tissues involved in hemocytes synthesis. In consequence, L. santolla hemocytes were more affected by the nutritional condition of crabs than by the DO level. Similar ndings have also been reported for Nephrops norvegicus during aerial exposure (Ridgway et al. 2006). Since immune defense largely relies on several hemocyte functions, such as coagulation, phagocytosis, encapsulation, and wound healing (Bachere 2000; Johansson et al. 2000; Pascual et al. 2004b), total hemocytes count is now also suggested as a reliable indicator of stress for L. santolla. The effect of DO on the nutritional condition of crab and on L. santolla hemocytes probably reects an immune suppression that might provide a critical window for opportunistic pathogens to become established when crabs are exposed to hypoxic conditions in the deep ocean or in culture conditions.
Acknowledgments This work was supported by FONDEF D05I10217, PBCT ACL 34, and DID S-2003-45. The authors want to express their special thanks to two anonymous referees who contributed to improve the manuscript. The experiments comply with the current Chilean animal care and manipulation legislation.

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