Fish Physiol Biochem (2016) 42:869–882 DOI 10.1007/s10695-015-0181-3

Aerobic and anaerobic enzymatic activity of orange roughy (Hoplostethus atlanticus) and ( splendens) from the Juan Fernandez area

L. M. Saavedra . R. A. Quin˜ones . R. R. Gonzalez-Saldı´a . E. J. Niklitschek

Received: 8 September 2015 / Accepted: 10 December 2015 / Published online: 19 December 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract The aerobic and anaerobic enzymatic with that migrate through OMZs. This poten- activity of two important commercial bathypelagic tial and the higher muscle citrate synthase and electron species living in the Juan Ferna´ndez seamounts was transport system activities indicate that alfonsino has analyzed: alfonsino (Beryx splendens) and orange greater swimming activity level than orange roughy. roughy (Hoplostethus atlanticus). These seamounts This species has also a high MDH/LDH ratio in its are influenced by the presence of an oxygen minimum heart, brain and liver, revealing a potential capacity to zone (OMZ) located between 160 and 250 m depth. conduct aerobic metabolism in these organs under Both species have vertical segregation; alfonsino is prolonged periods of environmental low oxygen able to stay in the OMZ, while orange roughy remains conditions, preventing lactic acid accumulation. With at greater depths. In this study, we compare the aerobic these metabolic characteristics, alfonsino may have and anaerobic capacity of these species, measuring the increased swimming activity to migrate and also could activity of key metabolic enzymes in different body stay for a period of time in the OMZ. The observed tissues (muscle, heart, brain and liver). Alfonsino has differences between alfonsino and orange roughy with higher anaerobic potential in its white muscle due to respect to their aerobic and anaerobic enzymatic greater lactate dehydrogenase (LDH) activity activity are consistent with their characteristic vertical (190.2 lmol NADH min-1 gww-1), which is related distributions and feeding behaviors. to its smaller body size, but it is also a feature shared

L. M. Saavedra (&) R. R. Gonzalez-Saldı´a Center for the Study of Multiple-Drivers on Marine Socio- Unidad de Biotecnologı´a Marina, Facultad de Ciencias Ecological Systems (MUSELS), Universidad de Naturales y Oceanogra´ficas, Universidad de Concepcio´n, Concepcio´n, Barrio Universitario S/N, Concepcio´n, Chile Casilla 160C, Concepcio´n, Chile e-mail: [email protected] E. J. Niklitschek R. A. Quin˜ones Centro i*mar, Universidad de Los Lagos, Camino a Interdisciplinary Center for Aquaculture Research Chinquihue Km 6, Casilla 557, Puerto Montt, ChileX (INCAR), Universidad de Concepcio´n, O’Higgins 1695, Regio´n Concepcio´n, Chile

R. A. Quin˜ones Departamento de Oceanografı´a, Facultad de Ciencias Naturales y Oceanogra´ficas, Universidad de Concepcio´n, Casilla 160C, Concepcio´n, Chile 123 870 Physiol Biochem (2016) 42:869–882

Keywords Seamounts Á Enzymatic activity Á roughy is bathypelagic and lives below the OMZ at Oxygen minimum zone Á Beryx splendens Á preferred depths of 500–1000 m, whereas alfonsino Hoplostethus atlanticus uses shallower habitats, with greater presence around 400 m (Niklitschek et al. 2007; Guerrero and Arana 2009) and daily migrations into more superficial waters (Vinnichenko 1997), entering and crossing Introduction the OMZ on a daily basis (Fig. 1). This vertical segregation between the habitats used Seamounts are highly productive ecosystems in which by the species should be reflected in important the upwelling of nutrient-rich water and the trapping of differences in their metabolic properties (Siebenaller diurnally migrating plankton (Rogers 1994) provide a and Somero 1989). For instance, interspecific com- unique deep-sea environment for fishes and invertebrates parisons of enzymatic activity have shown a general (Koslow 1997). In spite of the large number of seamounts pattern of decreasing metabolic activity with increas- in the global ocean, especially in the Pacific, relatively ing depth (Siebenaller and Somero 1989; Childress few studies have been carried out on the biology and and Thuesen 1992). Moreover, vertically migratory ecology of biota (Clark et al. 2014). fishes such as alfonsino are expected to have metabolic A stable and persistent feature of seamounts located adaptations that allow them to cross and remain in the in the eastern Pacific is the presence of a permanent OMZ (Childress and Seibel 1998), as this zone is oxygen minimum zone (OMZ) (Rabalais et al. 2010). known to be an important barrier for the distribution of Therefore fish inhabiting or crossing this layer as part marine organisms (White 1987; Eissler and Quin˜ones of their daily routine are expected to exhibit metabolic 1999; Gonza´lez and Quin˜ones 2002). Adaptations to and physiological adaptations, which are still poorly low oxygen availability in biota dwelling permanently understood (Martı´nez et al. 2011). This understanding or semipermanently in OMZ may be achieved using has become more relevant today when the increment several strategies, such as (1) more effective oxygen in hypoxic zones in the ocean worldwide has become a incorporation, (2) less metabolic demand and (3) global issue (Zhang et al. 2010; Diaz and Rosenberg conducting anaerobic metabolism (Childress and 2008; Rabalais et al. 2010), which due to global Seibel 1998). It has also been suggested that some warming could become more acute in the future (Justic vertically migrating species can alternate between et al. 1996; Diaz and Rosenberg 2008; Hansen and anaerobic metabolism while in the OMZ and aerobic Bendtsen 2009; Falkowski et al. 2011). metabolism when they encounter more oxygenated An important group of Eastern Pacific seamounts waters (Childress 1977). An important enzymatic corresponds to the Juan Fernandez Ridge, located on adaptation to hypoxia is the change in affinity of the Nazca Plate off the coast of Chile between enzymes involved in glycolysis and other pathways of 32–34°S and 73–82°W (Pilger 1981). Here the OMZ carbohydrate metabolism (Panepucci et al. 2000;Wu is composed of equatorial subsurface waters (Ahu- 2002; Pollock et al. 2007). mada and Chuecas 1979), ranges between 150 and Analysis of enzyme activities can be used as an 350 m depth and presents dissolved oxygen values approach to assess reliance on anaerobic metabolism -1 between 0.3 and 2.0 mL O2 L (Fig. 1a) (Nikl- in OMZ species (Childress and Seibel 1998; Yang itschek et al. 2007). Below this depth, oxygen levels et al. 1992), as well as to determine metabolic increase to a maximum of approximately 4 mL O2 - differences between fish that have different patterns L-1 around 600 m and decrease again to about of vertical distribution (Childress and Somero 1979; -1 2mLO2 L at a depth of approximately 950 m Siebenaller et al. 1982; Vetter et al. 1994; Childress (Chiang and Quin˜ones 2007; Niklitschek et al. 2007). 1995; Vetter and Lynn 1997). For, instance, citrate Two commercially important bathypelagic species synthase (CS), associated with oxidative phosphory- are exploited in this ridge, orange roughy [Ho- lation, is used as an indicator of aerobic metabolism, plostethus atlanticus (Collet)] and alfonsino [Beryx and lactate dehydrogenase (LDH) is indicative of splendens (Lowe)], which exhibit clear vertical seg- anaerobic metabolism (Childress 1995; Farwell et al. regation with only a slight overlap between 550 and 2007; Martı´nez et al. 2011). Malate dehydrogenase 650 m (Niklitschek et al. 2007, Fig. 1b, c). Orange (MDH) plays a role in both aerobic and anaerobic 123 Fish Physiol Biochem (2016) 42:869–882 871

Fig. 1 a Dissolved oxygen profile from a zonal transect (75°W to 78.8°W) of Juan Fernandez seamounts; b vertical distribution of alfonsino and c orange roughy in these seamounts (extracted from Niklitschek et al. 2007) pathways (Vetter et al. 1994), because the mitochon- respiration potential (Ikeda et al. 2006). Thus we drial isozyme (m-MDH) is a component of the Krebs characterize metabolic differences between species cycle and also passes reduced equivalents between the and explore hypotheses about the value of such mitochondria and the cytoplasm. The cytoplasmic differences as adaptive mechanisms making possible isozyme (s-MDH) shares the mentioned function with fairly distinct habitat use patterns under such strong m-MDH and in certain species can be important for gradients in hypoxic conditions. maintaining the cytoplasmatic redox balance during intense anaerobiosis (Hochachka and Somero 1984). In this paper, we analyze and compare the activity Materials and methods of different metabolic enzymes in several tissues of H. Atlanticus and B. Splendens collected at the Juan Collection and preservation of study Fernandez Ridge. We also estimate and compare metabolic rates through the activity of the electron Fish samples were obtained from deep-bottom trawls transport system (ETS), which is a measure of deployed between July 30 and August 7, 2005, by the

123 872 Fish Physiol Biochem (2016) 42:869–882

Fig. 2 Map showing the sampling sites. The three seamounts (JF2, JF3 and JF4) belong to the Juan Fernandez Archipelago. Coordinates correspond to UTM zone 18S

factory vessel ‘‘Betanzos,’’ as part of the 2005 annual samples were collected between 350 and 450 m from orange roughy–alfonsino hydroacoustic survey (Nik- JF2 (Table 1; Fig. 2). These sampling depth ranges litschek et al. 2006). The sampling area included three represented frequent catch strata for these two species seamounts (JF2, JF3 and JF4), all in the Juan in the study area. Fernandez Ridge area (Fig. 2). A total of 24 orange Once caught, each specimen was quickly sized roughy samples were obtained between 714 and (fork length, FL) and sections of white muscle, liver, 1178 m from seamounts JF3 and JF4; 10 alfonsino brain and heart were extracted and immediately stored

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Table 1 Classifications, depth range, mean weight and capture location of sampled individuals per species Specie Class Family Sampling Depth Average location range (m) weight (g)

Hoplostethus atlanticus Trachichthyidae JF3, JF4 400–1800 2080 Beryx splendens Actinopterygii Beryciformes JF2 30–850 820 in liquid nitrogen (-190 °C) for further enzymatic MDH activity (from oxaloacetate to malate) was analysis. The rest of each fish was kept on ice until measured in all tissues using the procedure described they were weighed in the laboratory on land. by Childress and Somero (1979) and Vetter et al. (1994). Homogenization Determination of enzymatic activities involved Tissue samples were thawed, weighed and homoge- in aerobic metabolism nized in 200 mM K2HPO4 buffer (pH 7.9), 0.3 % polyvinylpyrrolidone (PVP), 5 mM EDTA and 0.1 % The activity of the citrate synthase (CS) enzyme was Triton X-100, using an Ultra Turrax homogenizer in measured using a modified version of the method an ice bath. The amount of buffer solution that was proposed by Childress and Somero (1979) and Vetter added to each sample was calculated using a dilution et al. (1994). The reaction mixture contained 50 mM factor that varied between species and tissues: orange Imidazol/HCl pH 8.0 at 20 °C, 1.5 mM MgSO , roughy = 1:40 for muscle and brain and 1:100 for 4 0.1 mM acid, 5.5 mM dithiobis (2-nitrobenzoic) liver and heart; alfonsino = 1:67 for liver and brain (DTNB) and 0.06 mM acetyl-CoA. The supernatant and 1:100 for muscle and heart. After centrifugation of was added, and the mixture was incubated for 20 min the homogenate for 5 min at 3000 g (4 °C), one part of at room temperature. Then 0.2 mM oxalacetate was the supernatant was used for electron transport system added, and absorbance was measured. Absorbance (ETS) analysis and the rest for measuring enzymatic was determined at 412 nm. All determinations were activity. corrected using a blank containing the supernatant but in absence of oxalacetate. Determination of enzymatic activities involved in anaerobic metabolism Electron transport system (ETS) activity Measurements of the various enzyme activities were conducted by spectrophotometry and run in triplicate. ETS was estimated using the technique described by Assay temperature was between 15 and 16 °C. Lactate Packard (1971). This is an indirect enzymatic method pathway activity (LDH) was analyzed as representa- used to estimate the rate of oxygen consumption as an tive of anaerobic metabolism. The lactate pathway expression of the maximum potential activity of the maintains its metabolic rate under hypoxic environ- electron transporters in the respiratory chain at a mental or physiological conditions (Livingstone mitochondrial level. 1983). In situ ETS activity was calculated with the The assay mixture was modified from Schiedek Arrhenius equation (S = A exp (Ea/k (1/Ta - 1/Ts))),

(1997); it contained 80 mM K2HPO4 buffer (pH 7.9) where S is the in situ ETS, A is the ETS calculated at the and 3.2 mM pyruvate. Before measuring, 0.2 mM assay incubation temperature, Ea is the Arrhenius NADH was added to the mixture. Finally, an aliquot of activation energy, k is the gas constant -1 -1 supernatant was added and the decay of the NADH (1.987 cal mol deg ), Ta is the assay temperature absorption at 340 nm was measured. All the enzy- (°K) and Ts is the in situ temperature (°K). The matic activities were corrected for nonspecific NADH activation energy used was 16.2 (kcal mol-1) (Arı´s- oxidation. tegui and Montero 1995). The in situ temperature was

123 874 Fish Physiol Biochem (2016) 42:869–882 obtained from CTD data collected during the cruise MDH was greater in alfonsino than in orange (Niklitschek et al. 2006). roughy for all tissues analyzed (p \ 0.05) (Fig. 3b). All potential activities were expressed as wet ANCOVA analysis confirmed significant differences weight-specific activities. Thus the unit used to between species and showed no evidence of significant express LDH and MDH activity was lmol NADH body mass effects upon MDH activity in white muscle min-1 g-1wet weight (ww), CS activity was (Table 3; Fig. 4b). expressed as lmol DTNB min-1 g -1, and ETS as White muscle exhibited greater LDH than MDH -1 -1 lLO2 h g . activity in both species (Fig. 5). In all other tissues, MDH activity was higher than LDH activity. The Statistical analysis MDH/LDH ratio exhibited a similar pattern in both species, being lower in the muscle, intermediate in the One-way analysis of variance (ANOVA) was used to heart and higher in the brain (Table 4; Fig. 6). It evaluate differences in enzymatic activity between should be noted that this ratio was higher in all species, within tissues and between tissues within alfonsino tissues than in orange roughy tissues. species. Analysis of covariance (ANCOVA) was used Liver tissue showed a very high MDH/LDH ratio in to evaluate species and body mass effects on the alfonsino which was related to very low LDH activity, differences between enzymatic activities in fishes. while in orange roughy liver MDH/LDH ratio could not be determined due to the absence of detectable LDH activity in this tissue. Results CS LDH Considering all tissues, CS activity ranged between Considering all tissues analyzed, LDH activity ranged 0.55 and 3.3 lmol DTNB min-1 gww-1 in orange between 50.4–153 lmol NADH min-1 gww-1 in roughy and 3.31–14 lmol DTNB min-1 gww-1 in orange roughy and 4.1–190.2 lmol NADH min-1 - alfonsino (Table 2; Fig. 3c). gww-1 in alfonsino (Table 2; Fig. 3a). The activity of this enzyme was greater in almost all LDH activity in white muscle was significantly tissues of alfonsino compared with those of orange greater in alfonsino than in orange roughy (p B 0.01), roughy, with significant differences (p \ 0.05) in while all other tissues showed similar enzymatic activ- muscle, heart and brain (Fig. 3c). ities between species (Fig. 3a). Between tissues, within Orange roughy presented similar CS activity in species, orange roughy showed more LDH activity only heart, brain and liver tissues; all these were greater in the heart, whereas in alfonsino the activity of this than white muscle. Alfonsino showed very high CS enzyme was greater in both white muscle and heart. In activity in its heart, which was greater than in all other both species, LDH activity was rather low in the brain alfonsino tissues and significantly greater than in and very low or null in the liver (Table 2). ANCOVA orange roughy white muscle (p \ 0.001) (Fig. 3c). analysis confirmed significant differences between ANCOVA analysis confirmed significant differences species and showed that only for LDH in muscle the between species, but showed no evidence of signifi- differences between both species are size related, with cant body mass effects upon CS activity in white lower activity in heavier fishes (Table 3;Fig.4a). muscle (Table 3; Fig. 4b). MDH ETS Considering all tissues analyzed, MDH activity ranged between 10.3 and 236 lmol NADH min-1 gww-1 in Considering all tissues analyzed, ETS activity ranged -1 -1 -1 -1 orange roughy and 87–519 lmol NADH min gww between 9.76–1261.4 llO2 h gww in orange -1 -1 in alfonsino (Table 2;Fig.3b). This enzyme showed roughy and 236–1657 llO2 h gww in alfonsino lower activity than LDH in white muscle for both species. (Table 2; Fig. 3d).

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Table 2 Mean enzymatic activities in tissues of three different bathypelagic teleosts (±S.D) Weight (g) n Muscle n Heart n Brain n Liver

H. atlanticus LDH 2042 25 66.2 ± 26 18 153.1 ± 85 22 50.423 ± 12 21 n.d MDH 2042 24 10.3 ± 3.5 18 180 ± 88 21 170.3 ± 32 21 236 ± 57.4 ETS in situ 2067 23 46 ± 27.1 18 506 ± 276 19 652 ± 150 17 624.1 ± 146.8 CS 2055 23 0.55 ± 0.2 18 2.76 ± 1.1 19 3.3 ± 0.7 17 2.62 ± 0.5 B. splendens LDH 820 10 190.2 ± 62 10 144.2 ± 73 10 41.4 ± 7 8 4.1 ± 1.3 MDH 820 9 87 ± 18 10 519.3 ± 196 10 267.3 ± 39 8 337 ± 67 ETS in situ 820 8 236.2 ± 60 7 1657 ± 492 6 712 ± 130.6 4 803 ± 222 CS 820 9 3.31 ± 0.66 9 14 ± 5 9 6.2 ± 1.6 7 3.32 ± 0.5 Units of activity are in lmoles NADH min-1 gww-1 for LDH and MDH, in lmoles DTNB min-1 gww-1 for CS and in -1 -1 llO2 h gww for ETS

Fig. 3 Activities of a LDH, b MDH, c CS and d ETS in different tissues of orange roughy Hoplostethus atlanticus and alfonsino Beryx splendens. Significant differences between species indicated with asterisks (*p \ 0.05), (**p \ 0.001)

As for CS and MDH, the activity of ETS was lower significantly greater than the level observed in orange in white muscle from both species, and highest in roughy heart tissue (Fig. 3d). ANCOVA confirmed alfonsino heart tissue. This ETS activity was, in fact, significant differences in ETS activity in white muscle

123 876 Fish Physiol Biochem (2016) 42:869–882

Table 3 Marginal analyses Effect Sum of squares DF Fpvalue (p [ F) of covariance (Type II) to evaluate the effects of LDH muscle (R2 = 0.58) species, body mass and their Species 0.04302 1 2.1823 [0.1 interaction upon LDH, MDH, CS and ETS activity log10 (body mass) 0.13221 1 6.7073 <0.05 in white muscle Species 9 log10 (body mass) 0.01119 1 0.5676 [0.4 Residuals 0.59134 30 MDH muscle (R2 = 0.94) Species 0.99528 1 85.7131 <0.0001

log10 (body mass) 0.00127 1 0.1095 [0.7

Species 9 log10 (body mass) 0.01774 1 1.5274 [0.2 Residuals 0.31352 27 CS muscle (R2 = 0.94) Species 0.44812 1 45.5447 <0.0001

log10 (body mass) 0.07723 1 7.8494 <0.01

Species 9 log10 (body mass) 0.00533 1 0.5422 [0.4 Residuals 0.26566 27 ETS muscle (R2 = 0.63) Species 1.09827 1 15.7797 <0.001

log10 (body mass) 0.05264 1 0.7563 [0.3

Species 9 log10 (body mass) 0.03210 1 0.4613 [0.5 Bold font highlights Residuals 1.87920 27 significant effects between species, but showed no relationship to body their (1) habitat depth (*250 m between distribution weight (Fig. 4d). modes) and oxygen availability (*2mgl-1); and (2) Considering the enzyme activity of the electron behavior, including predation and presence/absence of transfer system to be a measure of respiration potential daily vertical migration. (Ikeda 1996; Ikeda et al. 2006), a positive correlation The higher levels of LDH activity we found in was observed between enzymatic activity and respi- alfonsino white muscle seem to be mainly related to a ration rate considering the species together, indicating body mass effect. This was not, however, the case of a linear increment in enzyme activity with higher MDH, CS and ETS, whose higher activity in several respiration rate (Fig. 7). alfonsino tissues exceeded differences due to potential body mass effects and was consistent with the general expectation of greater activity of metabolic enzymes Discussion in shallower species (Childress and Somero 1979; Somero 1992; Siebenaller et al. 1982; Childress 1995; Seamounts are relatively common features on the Vetter et al. 1994; Vetter and Lynn 1997; Drazen and ocean floor, which concentrate high ecological, eco- Seibel 2007). Nonetheless, due to our sampling design nomical and scientific interest. However, relatively (i.e., limited sampling opportunities), it was not few studies have been performed on the ecology of possible to separate species effects from depth these ecosystems and the biology of their biota. This effects.LDH activity was very similar between both study focused on some metabolic characteristics in species for heart, brain and liver, while in white two of the most abundant fishes inhabiting seamounts muscle, LDH showed the expected tendency for depth in the eastern South Pacific and elsewhere, finding inhabiting fishes (Vetter et al. 1994), with a higher clear differences in enzymatic activities involved in anaerobic activity in smaller fishes (alfonsino). The anaerobic and aerobic metabolism. These differences higher CS levels found in all alfonsino tissues are were consistent with expectations derived from known indicating a higher aerobic potential of this fish, which differences between these two species with respect to is consistent with his increased swimming capacity, 123 Fish Physiol Biochem (2016) 42:869–882 877

Fig. 4 Relationship between enzymatic activity in white expected values, represented as YALF and YOR, for alfonsino and muscle and body mass for a LDH, b MDH, c CS and d ETS orange roughy, respectively, correspond to the best model, in alfonsino [(open circle) Beryx splendens] and orange roughy selected for each enzyme using a stepwise backwards procedure [(filled circle) Hoplostethus atlanticus]. Regression lines and (alpha = 0.05) including prolonged burst swimming (Niklitschek particularly in alfonsino, indicate enhanced capacity et al. 2007). to maintain cytoplasmatic redox balance during High MDH/LDH ratios may cause an attenuated intense anaerobiosis, suggesting evolutionary adapta- pyruvate to lactate flux. As a consequence, carbohy- tion to protect these important organs under hypoxic drate metabolism will be largely channeled toward and anoxic conditions (Shapiro and Bobkova 1975; complete oxidation (Almeida-Val and Hochachka Panepucci et al. 2000; Panepucci et al. 2001). In 1993). Therefore, the MDH/LDH ratios much [1we contrast, the low MDH/LDH ratios we found in white observed in heart and brain tissues in both species, muscle tissue of both species suggest efficient 123 878 Fish Physiol Biochem (2016) 42:869–882

Fig. 6 MDH/LDH ratios in different tissues off H. atlanticus and B. splendens. Significant difference between species indicated with an asterisk (p \ 0.05)

roughy, allowing him to cross and to stay in the OMZ (Torres et al. 2012). In terms of aerobic enzymatic activity, white muscle showed the lowest levels of CS and ETS activity in both species, which was expected because of the physiology of this tissue (Somero and Childress 1980). This reaffirms the predominance of aerobic Fig. 5 Differences between lactate dehydrogenase (LDH) and metabolism in all analyzed tissues except the muscle. malate dehydrogenase (MDH) activity in different tissues from The white muscle, heart and brain tissues showed a alfonsino (B. splendens) and b orange roughy (H. atlanticus). Values are mean ± SD. Significant differences between significantly higher CS activity in alfonsino than in enzymes are given by *(p \ 0.01), ***(p \ 0.0001) orange roughy, which is consistent with higher MDH levels in these tissues and could be associated with greater capacity for sustained swimming, even under Table 4 Ratios of MDH/LDH activity for two bathypelagic species low oxygen conditions (Sullivan and Somero 1980). Moreover, observed differences in ETS activity in n MDH/LDH white muscle would be indicative of a greater Muscle Heart Brain respiratory rate in alfonsino (Ikeda 1996), which would be consistent with a more active feeding H. atlanticus 20 0.181 (0.12) 1.16 (0.3) 3.47 (0.6) strategy and a higher habitat temperature. Considering B. splendens 10 0.518 (0.2) 3.4 (0.9) 6.63 (1.4) the enzyme activity of the electron transfer system to Data are expressed as mean and S.E.M be a measure of respiration potential (Ikeda 1996; Ikeda et al. 2006), the very strong correlations found between muscle enzymatic activities and whole fish anaerobic production of ATP in this tissue regardless respiration rate (expressed as ETS) suggest that the of oxygen availability in the water column or the lower respiratory rate estimated for orange roughy is plasma. These results agree with those of Panepucci principally due to reduced potential for enzymatic et al. (2000), who found high MDH/LDH ratio in heart activity in its white muscle. This correlation is also in and brain of Rhinelepis strigosas exposed to different agreement with the results of Childress and Somero oxygen levels, showing the importance of these organs (1979), which showed that muscle enzymatic activity for the survival of this species when subjected to may provide a valid estimate of the VO2 of fish that extreme hypoxic conditions. The higher MDH/LDH cannot be recovered alive (e.g., fishes that have swim ratios observed in alfonsino for almost all tissues bladders and embolize during transit to the surface). provide support to the idea that alfonsino is more All previous results about enhanced tolerance to adapted to withstand hypoxic conditions than orange hypoxia and increased metabolic rate in alfonsino are 123 Fish Physiol Biochem (2016) 42:869–882 879

Fig. 7 Correlation between enzymatic activities and respiration rate measured as -1 -1 ETS (llO2 h g ). (open circle) Orange roughy and (filled circle) alfonsino

also consistent with observations indicating that this 1000 m in the ocean and declines exponentially with specie is capable to conduct daily feeding migrations depth (Warrant and Locket 2004). Thus, the lower connected to the daily migrations of its prey, mainly metabolic activity of orange roughy might be partially euphausiids and (Vinnichenko 1997; explained by a reduced ‘‘visual interaction’’ with its Alabsi 2011). To conduct this vertical migration, prey. This hypothesis, proposed by Childress (1995), alfonsino would take advantage of the previously predicts a rapid decline in metabolic rate with increas- described ability to cross the OMZ, which is a ing depth in deep-living animals with developed visual pervasive feature of the water column in the study perception systems (e.g., eyes). This would be due to area (Niklitschek et al. 2007). However, this hypoth- the effects of downward light diminishing upon prey– esis remains to be tested through direct behavioral predator reaction distance, resulting in a progressive studies in the Juan Fernandez seamounts area. reduction in energy expenditure with increasing depth We found a rather weak relationship between (Ikeda et al. 2006). Another important factor that might enzymatic activity and body weight for most enzymes be affecting the metabolic rate of these two species is and tissues, where significant body mass effects were temperature (Yang et al. 1992). However, since orange found only for LDH and CS in white muscle and for roughy lives at 2–6 °C and alfonsino lives between 6 MDH in brain tissue (not shown in our results). Both and 11 °C (Niklitshek et al. 2007), the difference enzymes (LDH and CS) showed a decrease in activity between the average temperatures where these species with increasing body weight, which is consistent with inhabit is not enough (4 °C in average) to explain any Vetter et al. (1994) who found that enzymatic effect on their metabolic rate. activities decreased with increasing body size in The greater activity of metabolic enzymes observed flatfishes inhabiting the continental slope ([400 m), in alfonsino contradicts the expectation that reduced presumably reflecting lower activity and growth in the metabolic rates may represent suitable adaptations to deep-living adult population. However, it is impossi- confront low oxygen levels in the OMZ (Childress and ble to make any sound inference about allometry from Seibel 1998), providing evidence that an enhanced our study due to the similarity in the body sizes of the glycolytic power may instead be the adaptive mech- organisms caught; a larger sample size and a wider anism or phenotypic response used by some fishes body mass range would be required to be more inhabiting or visiting the OMZ on a regular basis. This conclusive about these relationships. result is similar to that of Yang and Somero (1993), Although we could not demonstrate that the differ- who found a much higher LDH activity in red muscle ences observed in enzymatic activity between the of Sebastolobus alascanus living in the OMZ, which species are due to habitat differences, we hypothesize they interpreted as a phenotypic response to habitat that vertical distribution (and therefore pressure) plays hypoxia. As in the present study, they found no a significant role in metabolism. In fact, orange roughy evidence of greater anaerobic poise in the brain of lives below 600 m, with modes at 650 and 850 m, OMZ species than in the brain of species inhabiting while alfonsino remains above 500 m, with a strong shallower waters. The same trend was reported by mode at 450 m. Daylight only penetrates the upper Torres et al. (2012) comparing fish of systems with

123 880 Fish Physiol Biochem (2016) 42:869–882 different oxygen levels, where Arabic Sea species had Center for Aquaculture Research (INCAR; FONDAP 1511002) much greater LDH activity than fish from the Gulf of and FONDECYT 3150392. Mexico. It is important to note that the biochemical indicators found in the present study are not sufficient Compliance with ethical standards to establish with certainty that alfonsino is particularly adapted to crossing or visiting the OMZ. In fact, as Conflict of interest The authors declare that they have no mentioned before, the greater anaerobic and aerobic conflict of interest. capacity observed in its white muscle and the enzy- Research involving human participants and/or ani- matic protection of its vital organs against hypoxia may mals The fishes used in this research were collected from be just primary responses or adaptations that allow deep-bottom trawls deployed by the factory vessel ‘‘Betanzos,’’ increased swimming activity, related to an active as part of the 2005 annual orange roughy–alfonsino hydroa- coustic survey. The specimens were obtained from the trawls. feeding behavior including its daily feeding migra- Subsequently they were anaesthetized using benzocaine. Once tions. To prove increased tolerance to habitat hypoxia, they were fully anesthetized, they were dissected and tissues and other indicators such as circulatory and morphological organs preserved in liquid nitrogen. The fish died due to the adaptations should be investigated and compared removal of the heart, a needed tissue for the experimental objective. We did not conduct experimental work with animals between species, including gill surface areas, ventila- alive. We only used tissues preserved in liquid nitrogen. In any tion rate and volumes and the affinity for oxygen of case, all our experimental procedures at the laboratory are in their respiratory proteins (Childress and Seibel 1998; agreement with the regulations of the Chilean National Com- Seibel et al. 1999). mission on Scientific and Technological Research (CONICYT), Ministry of Education, Chilean Government. In conclusion, alfonsino exhibited greater anaero- bic potential in its white muscle than orange roughy, a potential that seems shared with other species that migrate through OMZs elsewhere (Yang et al. 1992; References Vetter and Lynn 1997; Torres et al. 2012). This potential and the higher white muscle CS and ETS Ahumada R, Chuecas L (1979) Algunas caracterı´sticas 0 0 activities indicate that alfonsino also has a higher hidrogra´ficas de la Bahı´a Concepcio´n (36°40 S; 73°02 W) ya´reas adyacentes (Chile). Gayana 8:1–56 swimming activity level than orange roughy. More- Alabsi NM (2011) Studies on the behavior of a deep-water fish, over, this species has a high MDH/LDH ratio in its the (Beryx splendens) using micro data heart, brain and liver, revealing a greater potential to loggers. Master thesis University of Tokyo conduct aerobic metabolism in these organs under Almeida-Val VMF, Hochachka PW (1993) Hypoxia tolerance in Amazon fishes: status of an underexplored biological prolonged periods of fast swimming and/or environ- ‘‘goldmine’’. In: Hochachka PW, Lutz PL, Sick T, mental low oxygen conditions, preventing lactic acid Rosenthal M, Van den Thillart G (eds) Surviving hypoxia: accumulation. All these metabolic differences made it mechanisms of control and adaptation. CRC Press, Boca possible for alfonsino and probably not possible for Raton, pp 435–445 Arı´stegui J, Montero MF (1995) The relationship between orange roughy to migrate through and sometimes community respiration and ETS activity in the ocean. remain for a certain period of time in the OMZ. J Plankton Res 17:1563–1571 Chiang OE, Quin˜ones RA (2007) Relationship between viral Acknowledgments This research was funded by the and prokaryotic abundance on the Bajo O’Higgins 1 Sea- Interdisciplinary Center for Aquaculture Research (INCAR; mount (Eastern South Pacific, Chile). Sci Mar 71:37–46 FONDAP 1511002). Sampling was made possible as part of Childress JJ (1977) Effects of pressure, temperature and oxygen research Project No 2004–13 from the Fondo de Investigacio´n on the oxygen consumption rate of the midwater copepod Pesquera, Chile (Undersecretariat of Fisheries, Ministry of Gaussia princeps. Mar Biol 39:19–24 Economy, Chile). R. Gonzalez was funded by FONDECYT Childress JJ (1995) Are there physiological and biochemical 234568 (CONICYT, Chile), E. Niklitschek by INNOVA Chile adaptations of metabolism in deep-sea animals? Tree Grant No. 34567 and Luisa Saavedra by FONDECYT 3150392 10:30–36 and Center for the study of multiple-drivers on marine socio- Childress JJ, Seibel BA (1998) Life at stable low oxygen levels: ecological systems (MUSELS, IC120019). adaptations of animals to oceanic oxygen minimum layers. J Exp Biol 201:1223–1232 Funding This study was funded by Fondo de Investigacio´n Childress JJ, Somero GN (1979) Depth-related enzymic activ- Pesquera, Chile (Undersecretariat of Fisheries, Ministry of ities in muscle, brain and heart of deep-living pelagic Economy, Chile) research Project No. 2004–13, Interdisciplinary marine teleosts. Mar Biol 52:273–283

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