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 alfonsino (Beryx splendens)
from the Juan Fernandez seamounts area
L. M. Saavedra . R. A. Quiñones .
R. R. Gonzalez-Saldı́a . 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
activity of two important commercial bathypelagic
species living in the Juan Fernández seamounts was
analyzed: alfonsino (Beryx splendens) and orange
roughy (Hoplostethus atlanticus). These seamounts
are influenced by the presence of an oxygen minimum
zone (OMZ) located between 160 and 250 m depth.
Both species have vertical segregation; alfonsino is
able to stay in the OMZ, while orange roughy remains
at greater depths. In this study, we compare the aerobic
and anaerobic capacity of these species, measuring the
activity of key metabolic enzymes in different body
tissues (muscle, heart, brain and liver). Alfonsino has
higher anaerobic potential in its white muscle due to
greater lactate dehydrogenase (LDH) activity
(190.2 lmol NADH min-1 g ww-1), which is related
to its smaller body size, but it is also a feature shared
with species that migrate through OMZs. This potential and the higher muscle citrate synthase and electron
transport system activities indicate that alfonsino has
greater swimming activity level than orange roughy.
This species has also a high MDH/LDH ratio in its
heart, brain and liver, revealing a potential capacity to
conduct aerobic metabolism in these organs under
prolonged periods of environmental low oxygen
conditions, preventing lactic acid accumulation. With
these metabolic characteristics, alfonsino may have
increased swimming activity to migrate and also could
stay for a period of time in the OMZ. The observed
differences between alfonsino and orange roughy with
respect to their aerobic and anaerobic enzymatic
activity are consistent with their characteristic vertical
distributions and feeding behaviors.
L. M. Saavedra (&)
Center for the Study of Multiple-Drivers on Marine SocioEcological Systems (MUSELS), Universidad de
Concepción, Barrio Universitario S/N, Concepción, Chile
e-mail: [email protected]
R. R. Gonzalez-Saldı́a
Unidad de Biotecnologı́a Marina, Facultad de Ciencias
Naturales y Oceanográficas, Universidad de Concepción,
Casilla 160C, Concepción, Chile
R. A. Quiñones
Interdisciplinary Center for Aquaculture Research
(INCAR), Universidad de Concepción, O’Higgins 1695,
Concepción, Chile
E. J. Niklitschek
Centro i*mar, Universidad de Los Lagos, Camino a
Chinquihue Km 6, Casilla 557, Puerto Montt, ChileX
Región
R. A. Quiñones
Departamento de Oceanografı́a, Facultad de Ciencias
Naturales y Oceanográficas, Universidad de Concepción,
Casilla 160C, Concepción, Chile
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870
Keywords Seamounts Enzymatic activity Oxygen minimum zone Beryx splendens Hoplostethus atlanticus
Introduction
Seamounts are highly productive ecosystems in which
the upwelling of nutrient-rich water and the trapping of
diurnally migrating plankton (Rogers 1994) provide a
unique deep-sea environment for fishes and invertebrates
(Koslow 1997). In spite of the large number of seamounts
in the global ocean, especially in the Pacific, relatively
few studies have been carried out on the biology and
ecology of seamount biota (Clark et al. 2014).
A stable and persistent feature of seamounts located
in the eastern Pacific is the presence of a permanent
oxygen minimum zone (OMZ) (Rabalais et al. 2010).
Therefore fish inhabiting or crossing this layer as part
of their daily routine are expected to exhibit metabolic
and physiological adaptations, which are still poorly
understood (Martı́nez et al. 2011). This understanding
has become more relevant today when the increment
in hypoxic zones in the ocean worldwide has become a
global issue (Zhang et al. 2010; Diaz and Rosenberg
2008; Rabalais et al. 2010), which due to global
warming could become more acute in the future (Justic
et al. 1996; Diaz and Rosenberg 2008; Hansen and
Bendtsen 2009; Falkowski et al. 2011).
An important group of Eastern Pacific seamounts
corresponds to the Juan Fernandez Ridge, located on
the Nazca Plate off the coast of Chile between
32–34°S and 73–82°W (Pilger 1981). Here the OMZ
is composed of equatorial subsurface waters (Ahumada and Chuecas 1979), ranges between 150 and
350 m depth and presents dissolved oxygen values
between 0.3 and 2.0 mL O2 L-1 (Fig. 1a) (Niklitschek et al. 2007). Below this depth, oxygen levels
increase to a maximum of approximately 4 mL O2 L-1 around 600 m and decrease again to about
2 mL O2 L-1 at a depth of approximately 950 m
(Chiang and Quiñones 2007; Niklitschek et al. 2007).
Two commercially important bathypelagic species
are exploited in this ridge, orange roughy [Hoplostethus atlanticus (Collet)] and alfonsino [Beryx
splendens (Lowe)], which exhibit clear vertical segregation with only a slight overlap between 550 and
650 m (Niklitschek et al. 2007, Fig. 1b, c). Orange
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Fish Physiol Biochem (2016) 42:869–882
roughy is bathypelagic and lives below the OMZ at
preferred depths of 500–1000 m, whereas alfonsino
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
the OMZ on a daily basis (Fig. 1).
This vertical segregation between the habitats used
by the species should be reflected in important
differences in their metabolic properties (Siebenaller
and Somero 1989). For instance, interspecific comparisons of enzymatic activity have shown a general
pattern of decreasing metabolic activity with increasing depth (Siebenaller and Somero 1989; Childress
and Thuesen 1992). Moreover, vertically migratory
fishes such as alfonsino are expected to have metabolic
adaptations that allow them to cross and remain in the
OMZ (Childress and Seibel 1998), as this zone is
known to be an important barrier for the distribution of
marine organisms (White 1987; Eissler and Quiñones
1999; González and Quiñones 2002). Adaptations to
low oxygen availability in biota dwelling permanently
or semipermanently in OMZ may be achieved using
several strategies, such as (1) more effective oxygen
incorporation, (2) less metabolic demand and (3)
conducting anaerobic metabolism (Childress and
Seibel 1998). It has also been suggested that some
vertically migrating species can alternate between
anaerobic metabolism while in the OMZ and aerobic
metabolism when they encounter more oxygenated
waters (Childress 1977). An important enzymatic
adaptation to hypoxia is the change in affinity of
enzymes involved in glycolysis and other pathways of
carbohydrate metabolism (Panepucci et al. 2000; Wu
2002; Pollock et al. 2007).
Analysis of enzyme activities can be used as an
approach to assess reliance on anaerobic metabolism
in OMZ species (Childress and Seibel 1998; Yang
et al. 1992), as well as to determine metabolic
differences between fish that have different patterns
of vertical distribution (Childress and Somero 1979;
Siebenaller et al. 1982; Vetter et al. 1994; Childress
1995; Vetter and Lynn 1997). For, instance, citrate
synthase (CS), associated with oxidative phosphorylation, is used as an indicator of aerobic metabolism,
and lactate dehydrogenase (LDH) is indicative of
anaerobic metabolism (Childress 1995; Farwell et al.
2007; Martı́nez et al. 2011). Malate dehydrogenase
(MDH) plays a role in both aerobic and anaerobic
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 mitochondrial isozyme (m-MDH) is a component of the Krebs
cycle and also passes reduced equivalents between the
mitochondria and the cytoplasm. The cytoplasmic
isozyme (s-MDH) shares the mentioned function with
m-MDH and in certain species can be important for
maintaining the cytoplasmatic redox balance during
intense anaerobiosis (Hochachka and Somero 1984).
In this paper, we analyze and compare the activity
of different metabolic enzymes in several tissues of H.
Atlanticus and B. Splendens collected at the Juan
Fernandez Ridge. We also estimate and compare
metabolic rates through the activity of the electron
transport system (ETS), which is a measure of
respiration potential (Ikeda et al. 2006). Thus we
characterize metabolic differences between species
and explore hypotheses about the value of such
differences as adaptive mechanisms making possible
fairly distinct habitat use patterns under such strong
gradients in hypoxic conditions.
Materials and methods
Collection and preservation of study animals
Fish samples were obtained from deep-bottom trawls
deployed between July 30 and August 7, 2005, by the
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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
orange roughy–alfonsino hydroacoustic survey (Niklitschek et al. 2006). The sampling area included three
seamounts (JF2, JF3 and JF4), all in the Juan
Fernandez Ridge area (Fig. 2). A total of 24 orange
roughy samples were obtained between 714 and
1178 m from seamounts JF3 and JF4; 10 alfonsino
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samples were collected between 350 and 450 m from
JF2 (Table 1; Fig. 2). These sampling depth ranges
represented frequent catch strata for these two species
in the study area.
Once caught, each specimen was quickly sized
(fork length, FL) and sections of white muscle, liver,
brain and heart were extracted and immediately stored
Fish Physiol Biochem (2016) 42:869–882
873
Table 1 Classifications, depth range, mean weight and capture location of sampled individuals per species
Specie
Class
Order
Family
Sampling
location
Depth
range (m)
Average
weight (g)
Hoplostethus atlanticus
Actinopterygii
Beryciformes
Trachichthyidae
JF3, JF4
400–1800
2080
Beryx splendens
Actinopterygii
Beryciformes
Berycidae
JF2
30–850
820
in liquid nitrogen (-190 °C) for further enzymatic
analysis. The rest of each fish was kept on ice until
they were weighed in the laboratory on land.
MDH activity (from oxaloacetate to malate) was
measured in all tissues using the procedure described
by Childress and Somero (1979) and Vetter et al.
(1994).
Homogenization
Tissue samples were thawed, weighed and homogenized in 200 mM K2HPO4 buffer (pH 7.9), 0.3 %
polyvinylpyrrolidone (PVP), 5 mM EDTA and 0.1 %
Triton X-100, using an Ultra Turrax homogenizer in
an ice bath. The amount of buffer solution that was
added to each sample was calculated using a dilution
factor that varied between species and tissues: orange
roughy = 1:40 for muscle and brain and 1:100 for
liver and heart; alfonsino = 1:67 for liver and brain
and 1:100 for muscle and heart. After centrifugation of
the homogenate for 5 min at 3000 g (4 °C), one part of
the supernatant was used for electron transport system
(ETS) analysis and the rest for measuring enzymatic
activity.
Determination of enzymatic activities involved
in anaerobic metabolism
Determination of enzymatic activities involved
in aerobic metabolism
The activity of the citrate synthase (CS) enzyme was
measured using a modified version of the method
proposed by Childress and Somero (1979) and Vetter
et al. (1994). The reaction mixture contained 50 mM
Imidazol/HCl pH 8.0 at 20 °C, 1.5 mM MgSO4,
0.1 mM acid, 5.5 mM dithiobis (2-nitrobenzoic)
(DTNB) and 0.06 mM acetyl-CoA. The supernatant
was added, and the mixture was incubated for 20 min
at room temperature. Then 0.2 mM oxalacetate was
added, and absorbance was measured. Absorbance
was determined at 412 nm. All determinations were
corrected using a blank containing the supernatant but
in absence of oxalacetate.
Electron transport system (ETS) activity
Measurements of the various enzyme activities were
conducted by spectrophotometry and run in triplicate.
Assay temperature was between 15 and 16 °C. Lactate
pathway activity (LDH) was analyzed as representative of anaerobic metabolism. The lactate pathway
maintains its metabolic rate under hypoxic environmental or physiological conditions (Livingstone
1983).
The assay mixture was modified from Schiedek
(1997); it contained 80 mM K2HPO4 buffer (pH 7.9)
and 3.2 mM pyruvate. Before measuring, 0.2 mM
NADH was added to the mixture. Finally, an aliquot of
supernatant was added and the decay of the NADH
absorption at 340 nm was measured. All the enzymatic activities were corrected for nonspecific NADH
oxidation.
ETS was estimated using the technique described by
Packard (1971). This is an indirect enzymatic method
used to estimate the rate of oxygen consumption as an
expression of the maximum potential activity of the
electron transporters in the respiratory chain at a
mitochondrial level.
In situ ETS activity was calculated with the
Arrhenius equation (S = A exp (Ea/k (1/Ta - 1/Ts))),
where S is the in situ ETS, A is the ETS calculated at the
assay incubation temperature, Ea is the Arrhenius
activation energy, k is the gas constant
(1.987 cal mol-1 deg-1), Ta is the assay temperature
(°K) and Ts is the in situ temperature (°K). The
activation energy used was 16.2 (kcal mol-1) (Arı́stegui and Montero 1995). The in situ temperature was
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874
obtained from CTD data collected during the cruise
(Niklitschek et al. 2006).
All potential activities were expressed as wet
weight-specific activities. Thus the unit used to
express LDH and MDH activity was lmol NADH
min-1 g-1wet weight (ww), CS activity was
expressed as lmol DTNB min-1 g -1, and ETS as
lL O2 h-1 g-1.
Statistical analysis
One-way analysis of variance (ANOVA) was used to
evaluate differences in enzymatic activity between
species, within tissues and between tissues within
species. Analysis of covariance (ANCOVA) was used
to evaluate species and body mass effects on the
differences between enzymatic activities in fishes.
Fish Physiol Biochem (2016) 42:869–882
MDH was greater in alfonsino than in orange
roughy for all tissues analyzed (p \ 0.05) (Fig. 3b).
ANCOVA analysis confirmed significant differences
between species and showed no evidence of significant
body mass effects upon MDH activity in white muscle
(Table 3; Fig. 4b).
White muscle exhibited greater LDH than MDH
activity in both species (Fig. 5). In all other tissues,
MDH activity was higher than LDH activity. The
MDH/LDH ratio exhibited a similar pattern in both
species, being lower in the muscle, intermediate in the
heart and higher in the brain (Table 4; Fig. 6). It
should be noted that this ratio was higher in all
alfonsino tissues than in orange roughy tissues.
Liver tissue showed a very high MDH/LDH ratio in
alfonsino which was related to very low LDH activity,
while in orange roughy liver MDH/LDH ratio could
not be determined due to the absence of
detectable LDH activity in this tissue.
Results
LDH
Considering all tissues analyzed, LDH activity ranged
between 50.4–153 lmol NADH min-1 g ww-1 in
orange roughy and 4.1–190.2 lmol NADH min-1 g ww-1 in alfonsino (Table 2; Fig. 3a).
LDH activity in white muscle was significantly
greater in alfonsino than in orange roughy (p B 0.01),
while all other tissues showed similar enzymatic activities between species (Fig. 3a). Between tissues, within
species, orange roughy showed more LDH activity only
in the heart, whereas in alfonsino the activity of this
enzyme was greater in both white muscle and heart. In
both species, LDH activity was rather low in the brain
and very low or null in the liver (Table 2). ANCOVA
analysis confirmed significant differences between
species and showed that only for LDH in muscle the
differences between both species are size related, with
lower activity in heavier fishes (Table 3; Fig. 4a).
CS
Considering all tissues, CS activity ranged between
0.55 and 3.3 lmol DTNB min-1 g ww-1 in orange
roughy and 3.31–14 lmol DTNB min-1 g ww-1 in
alfonsino (Table 2; Fig. 3c).
The activity of this enzyme was greater in almost all
tissues of alfonsino compared with those of orange
roughy, with significant differences (p \ 0.05) in
muscle, heart and brain (Fig. 3c).
Orange roughy presented similar CS activity in
heart, brain and liver tissues; all these were greater
than white muscle. Alfonsino showed very high CS
activity in its heart, which was greater than in all other
alfonsino tissues and significantly greater than in
orange roughy white muscle (p \ 0.001) (Fig. 3c).
ANCOVA analysis confirmed significant differences
between species, but showed no evidence of significant body mass effects upon CS activity in white
muscle (Table 3; Fig. 4b).
MDH
ETS
Considering all tissues analyzed, MDH activity ranged
between 10.3 and 236 lmol NADH min-1 g ww-1 in
orange roughy and 87–519 lmol NADH min-1 g ww-1
in alfonsino (Table 2; Fig. 3b). This enzyme showed
lower activity than LDH in white muscle for both species.
123
Considering all tissues analyzed, ETS activity ranged
between 9.76–1261.4 ll O2 h-1 g ww-1 in orange
roughy and 236–1657 ll O2 h-1 g ww-1 in alfonsino
(Table 2; Fig. 3d).
Fish Physiol Biochem (2016) 42:869–882
875
Table 2 Mean enzymatic activities in tissues of three different bathypelagic teleosts (±S.D)
Weight (g)
n
Muscle
n
Heart
153.1 ± 85
n
Brain
22
50.423 ± 12
n
Liver
H. atlanticus
LDH
2042
25
66.2 ± 26
18
21
n.d
MDH
ETS in situ
2042
2067
24
23
10.3 ± 3.5
46 ± 27.1
18
18
180 ± 88
506 ± 276
21
19
170.3 ± 32
652 ± 150
21
17
236 ± 57.4
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
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
236.2 ± 60
7
1657 ± 492
6
712 ± 130.6
4
803 ± 222
9
14 ± 5
9
6.2 ± 1.6
7
3.32 ± 0.5
B. splendens
ETS in situ
820
8
CS
820
9
3.31 ± 0.66
Units of activity are in lmoles NADH min-1 g ww-1 for LDH and MDH, in lmoles DTNB min-1 g ww-1 for CS and in
ll O2 h-1 g ww-1 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
in white muscle from both species, and highest in
alfonsino heart tissue. This ETS activity was, in fact,
significantly greater than the level observed in orange
roughy heart tissue (Fig. 3d). ANCOVA confirmed
significant differences in ETS activity in white muscle
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876
Table 3 Marginal analyses
of covariance (Type II) to
evaluate the effects of
species, body mass and their
interaction upon LDH,
MDH, CS and ETS activity
in white muscle
Fish Physiol Biochem (2016) 42:869–882
Effect
Sum of squares
DF
F
p value (p [ F)
LDH muscle (R2 = 0.58)
[0.1
Species
0.04302
1
2.1823
log10 (body mass)
0.13221
1
6.7073
<0.05
Species 9 log10 (body mass)
0.01119
1
0.5676
[0.4
Residuals
0.59134
30
Species
0.99528
1
85.7131
log10 (body mass)
Species 9 log10 (body mass)
0.00127
0.01774
1
1
0.1095
1.5274
Residuals
0.31352
27
Species
0.44812
1
45.5447
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
Species
1.09827
1
15.7797
log10 (body mass)
0.05264
1
0.7563
[0.3
Species 9 log10 (body mass)
0.03210
1
0.4613
[0.5
Residuals
1.87920
27
2
MDH muscle (R = 0.94)
<0.0001
[0.7
[0.2
2
CS muscle (R = 0.94)
<0.0001
ETS muscle (R2 = 0.63)
Bold font highlights
significant effects
between species, but showed no relationship to body
weight (Fig. 4d).
Considering the enzyme activity of the electron
transfer system to be a measure of respiration potential
(Ikeda 1996; Ikeda et al. 2006), a positive correlation
was observed between enzymatic activity and respiration rate considering the species together, indicating
a linear increment in enzyme activity with higher
respiration rate (Fig. 7).
Discussion
Seamounts are relatively common features on the
ocean floor, which concentrate high ecological, economical and scientific interest. However, relatively
few studies have been performed on the ecology of
these ecosystems and the biology of their biota. This
study focused on some metabolic characteristics in
two of the most abundant fishes inhabiting seamounts
in the eastern South Pacific and elsewhere, finding
clear differences in enzymatic activities involved in
anaerobic and aerobic metabolism. These differences
were consistent with expectations derived from known
differences between these two species with respect to
123
<0.001
their (1) habitat depth (*250 m between distribution
modes) and oxygen availability (*2 mg l-1); and (2)
behavior, including predation and presence/absence of
daily vertical migration.
The higher levels of LDH activity we found in
alfonsino white muscle seem to be mainly related to a
body mass effect. This was not, however, the case of
MDH, CS and ETS, whose higher activity in several
alfonsino tissues exceeded differences due to potential
body mass effects and was consistent with the general
expectation of greater activity of metabolic enzymes
in shallower species (Childress and Somero 1979;
Somero 1992; Siebenaller et al. 1982; Childress 1995;
Vetter et al. 1994; Vetter and Lynn 1997; Drazen and
Seibel 2007). Nonetheless, due to our sampling design
(i.e., limited sampling opportunities), it was not
possible to separate species effects from depth
effects.LDH activity was very similar between both
species for heart, brain and liver, while in white
muscle, LDH showed the expected tendency for depth
inhabiting fishes (Vetter et al. 1994), with a higher
anaerobic activity in smaller fishes (alfonsino). The
higher CS levels found in all alfonsino tissues are
indicating a higher aerobic potential of this fish, which
is consistent with his increased swimming capacity,
Fish Physiol Biochem (2016) 42:869–882
877
Fig. 4 Relationship between enzymatic activity in white
muscle and body mass for a LDH, b MDH, c CS and d ETS
in alfonsino [(open circle) Beryx splendens] and orange roughy
[(filled circle) Hoplostethus atlanticus]. Regression lines and
expected values, represented as YALF and YOR, for alfonsino and
orange roughy, respectively, correspond to the best model,
selected for each enzyme using a stepwise backwards procedure
(alpha = 0.05)
including prolonged burst swimming (Niklitschek
et al. 2007).
High MDH/LDH ratios may cause an attenuated
pyruvate to lactate flux. As a consequence, carbohydrate metabolism will be largely channeled toward
complete oxidation (Almeida-Val and Hochachka
1993). Therefore, the MDH/LDH ratios much [1 we
observed in heart and brain tissues in both species,
particularly in alfonsino, indicate enhanced capacity
to maintain cytoplasmatic redox balance during
intense anaerobiosis, suggesting evolutionary adaptation to protect these important organs under hypoxic
and anoxic conditions (Shapiro and Bobkova 1975;
Panepucci et al. 2000; Panepucci et al. 2001). In
contrast, the low MDH/LDH ratios we found in white
muscle tissue of both species suggest efficient
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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)
Fig. 5 Differences between lactate dehydrogenase (LDH) and
malate dehydrogenase (MDH) activity in different tissues from
a alfonsino (B. splendens) and b orange roughy (H. atlanticus).
Values are mean ± SD. Significant differences between
enzymes are given by *(p \ 0.01), ***(p \ 0.0001)
Table 4 Ratios of MDH/LDH activity for two bathypelagic
species
n
MDH/LDH
Muscle
Heart
Brain
H. atlanticus
20
0.181 (0.12)
1.16 (0.3)
3.47 (0.6)
B. splendens
10
0.518 (0.2)
3.4 (0.9)
6.63 (1.4)
Data are expressed as mean and S.E.M
anaerobic production of ATP in this tissue regardless
of oxygen availability in the water column or the
plasma. These results agree with those of Panepucci
et al. (2000), who found high MDH/LDH ratio in heart
and brain of Rhinelepis strigosas exposed to different
oxygen levels, showing the importance of these organs
for the survival of this species when subjected to
extreme hypoxic conditions. The higher MDH/LDH
ratios observed in alfonsino for almost all tissues
provide support to the idea that alfonsino is more
adapted to withstand hypoxic conditions than orange
123
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
metabolism in all analyzed tissues except the muscle.
The white muscle, heart and brain tissues showed
significantly higher CS activity in alfonsino than in
orange roughy, which is consistent with higher MDH
levels in these tissues and could be associated with
greater capacity for sustained swimming, even under
low oxygen conditions (Sullivan and Somero 1980).
Moreover, observed differences in ETS activity in
white muscle would be indicative of a greater
respiratory rate in alfonsino (Ikeda 1996), which
would be consistent with a more active feeding
strategy and a higher habitat temperature. Considering
the enzyme activity of the electron transfer system to
be a measure of respiration potential (Ikeda 1996;
Ikeda et al. 2006), the very strong correlations found
between muscle enzymatic activities and whole fish
respiration rate (expressed as ETS) suggest that the
lower respiratory rate estimated for orange roughy is
principally due to reduced potential for enzymatic
activity in its white muscle. This correlation is also in
agreement with the results of Childress and Somero
(1979), which showed that muscle enzymatic activity
may provide a valid estimate of the VO2 of fish that
cannot be recovered alive (e.g., fishes that have swim
bladders and embolize during transit to the surface).
All previous results about enhanced tolerance to
hypoxia and increased metabolic rate in alfonsino are
Fish Physiol Biochem (2016) 42:869–882
879
Fig. 7 Correlation between
enzymatic activities and
respiration rate measured as
ETS (ll O2 h-1 g-1). (open
circle) Orange roughy and
(filled circle) alfonsino
also consistent with observations indicating that this
specie is capable to conduct daily feeding migrations
connected to the daily migrations of its prey, mainly
euphausiids and cephalopods (Vinnichenko 1997;
Alabsi 2011). To conduct this vertical migration,
alfonsino would take advantage of the previously
described ability to cross the OMZ, which is a
pervasive feature of the water column in the study
area (Niklitschek et al. 2007). However, this hypothesis remains to be tested through direct behavioral
studies in the Juan Fernandez seamounts area.
We found a rather weak relationship between
enzymatic activity and body weight for most enzymes
and tissues, where significant body mass effects were
found only for LDH and CS in white muscle and for
MDH in brain tissue (not shown in our results). Both
enzymes (LDH and CS) showed a decrease in activity
with increasing body weight, which is consistent with
Vetter et al. (1994) who found that enzymatic
activities decreased with increasing body size in
flatfishes inhabiting the continental slope ([400 m),
presumably reflecting lower activity and growth in the
deep-living adult population. However, it is impossible to make any sound inference about allometry from
our study due to the similarity in the body sizes of the
organisms caught; a larger sample size and a wider
body mass range would be required to be more
conclusive about these relationships.
Although we could not demonstrate that the differences observed in enzymatic activity between the
species are due to habitat differences, we hypothesize
that vertical distribution (and therefore pressure) plays
a significant role in metabolism. In fact, orange roughy
lives below 600 m, with modes at 650 and 850 m,
while alfonsino remains above 500 m, with a strong
mode at 450 m. Daylight only penetrates the upper
1000 m in the ocean and declines exponentially with
depth (Warrant and Locket 2004). Thus, the lower
metabolic activity of orange roughy might be partially
explained by a reduced ‘‘visual interaction’’ with its
prey. This hypothesis, proposed by Childress (1995),
predicts a rapid decline in metabolic rate with increasing depth in deep-living animals with developed visual
perception systems (e.g., eyes). This would be due to
the effects of downward light diminishing upon prey–
predator reaction distance, resulting in a progressive
reduction in energy expenditure with increasing depth
(Ikeda et al. 2006). Another important factor that might
be affecting the metabolic rate of these two species is
temperature (Yang et al. 1992). However, since orange
roughy lives at 2–6 °C and alfonsino lives between 6
and 11 °C (Niklitshek et al. 2007), the difference
between the average temperatures where these species
inhabit is not enough (4 °C in average) to explain any
effect on their metabolic rate.
The greater activity of metabolic enzymes observed
in alfonsino contradicts the expectation that reduced
metabolic rates may represent suitable adaptations to
confront low oxygen levels in the OMZ (Childress and
Seibel 1998), providing evidence that an enhanced
glycolytic power may instead be the adaptive mechanism or phenotypic response used by some fishes
inhabiting or visiting the OMZ on a regular basis. This
result is similar to that of Yang and Somero (1993),
who found a much higher LDH activity in red muscle
of Sebastolobus alascanus living in the OMZ, which
they interpreted as a phenotypic response to habitat
hypoxia. As in the present study, they found no
evidence of greater anaerobic poise in the brain of
OMZ species than in the brain of species inhabiting
shallower waters. The same trend was reported by
Torres et al. (2012) comparing fish of systems with
123
880
different oxygen levels, where Arabic Sea species had
much greater LDH activity than fish from the Gulf of
Mexico. It is important to note that the biochemical
indicators found in the present study are not sufficient
to establish with certainty that alfonsino is particularly
adapted to crossing or visiting the OMZ. In fact, as
mentioned before, the greater anaerobic and aerobic
capacity observed in its white muscle and the enzymatic protection of its vital organs against hypoxia may
be just primary responses or adaptations that allow
increased swimming activity, related to an active
feeding behavior including its daily feeding migrations. To prove increased tolerance to habitat hypoxia,
other indicators such as circulatory and morphological
adaptations should be investigated and compared
between species, including gill surface areas, ventilation rate and volumes and the affinity for oxygen of
their respiratory proteins (Childress and Seibel 1998;
Seibel et al. 1999).
In conclusion, alfonsino exhibited greater anaerobic 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;
Vetter and Lynn 1997; Torres et al. 2012). This
potential and the higher white muscle CS and ETS
activities indicate that alfonsino also has a higher
swimming activity level than orange roughy. Moreover, this species has a high MDH/LDH ratio in its
heart, brain and liver, revealing a greater potential to
conduct aerobic metabolism in these organs under
prolonged periods of fast swimming and/or environmental low oxygen conditions, preventing lactic acid
accumulation. All these metabolic differences made it
possible for alfonsino and probably not possible for
orange roughy to migrate through and sometimes
remain for a certain period of time in the OMZ.
Acknowledgments This research was funded by the
Interdisciplinary Center for Aquaculture Research (INCAR;
FONDAP 1511002). Sampling was made possible as part of
research Project No 2004–13 from the Fondo de Investigación
Pesquera, Chile (Undersecretariat of Fisheries, Ministry of
Economy, Chile). R. Gonzalez was funded by FONDECYT
234568 (CONICYT, Chile), E. Niklitschek by INNOVA Chile
Grant No. 34567 and Luisa Saavedra by FONDECYT 3150392
and Center for the study of multiple-drivers on marine socioecological systems (MUSELS, IC120019).
Funding This study was funded by Fondo de Investigación
Pesquera, Chile (Undersecretariat of Fisheries, Ministry of
Economy, Chile) research Project No. 2004–13, Interdisciplinary
123
Fish Physiol Biochem (2016) 42:869–882
Center for Aquaculture Research (INCAR; FONDAP 1511002)
and FONDECYT 3150392.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
Research involving human participants and/or animals The fishes used in this research were collected from
deep-bottom trawls deployed by the factory vessel ‘‘Betanzos,’’
as part of the 2005 annual orange roughy–alfonsino hydroacoustic survey. The specimens were obtained from the trawls.
Subsequently they were anaesthetized using benzocaine. Once
they were fully anesthetized, they were dissected and tissues and
organs preserved in liquid nitrogen. The fish died due to the
removal of the heart, a needed tissue for the experimental
objective. We did not conduct experimental work with animals
alive. We only used tissues preserved in liquid nitrogen. In any
case, all our experimental procedures at the laboratory are in
agreement with the regulations of the Chilean National Commission on Scientific and Technological Research (CONICYT),
Ministry of Education, Chilean Government.
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