Post on 19-Feb-2023
ORIGINAL PAPER
Effect of dissolved oxygen level on respiratory metabolism,nutritional physiology, and immune condition of southern kingcrab Lithodes santolla (Molina, 1782) (Decapoda, Lithodidae)
Kurt Paschke Æ Juan Pablo Cumillaf Æ Sergio Loyola Æ Paulina Gebauer ÆMauricio Urbina Æ Marıa Eugenia Chimal Æ Cristina Pascual ÆCarlos 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 fluctuating oxygen regi-
mens. Physiological mechanisms enhance the ability of
these crustaceans to cope with acute episodes of hypoxia.
Southern king crab, Lithodes santolla, fishery is important
in the south of South America, and some data describe
fishing 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 consump-
tion rate; thereafter, blood oxyhemocyanin (Hc), protein
concentration, as well as hemocytes, were sampled. Freeze-
dried animals were dissected, and digestive gland metab-
olites (glycogen, protein, glucose, cholesterol, acylglyc-
erol, and lactate) and digestive enzyme activity (general
protease, trypsin, and chymotrypsin), as well as gill lactate
dehydrogenase (LDH) activity, were quantified. In the
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, cho-
lesterol, 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 quantified 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 patho-
gens 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 environ-
ment, particularly associated to upwelling events (Gran-
tham et al. 2004), as well as in shallow coastal zones.
Coastal hypoxia generally follows seasonal patterns, some
of them directly related to the influx of freshwater and
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
123
Mar Biol (2010) 157:7–18
DOI 10.1007/s00227-009-1291-1
anthropogenic eutrophication (Turner et al. 2008). Hypoxic
zones are becoming more widespread and are one of the
most deleterious human-induced impacts on benthic com-
munities (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 (Dıaz and
Rosenberg 1995; Grantham et al. 2004). Generally, toler-
ance levels are higher for organisms residing in the sedi-
ments, whereas mobile organisms, such as fish and
crustaceans, may exhibit behavioral responses to avoid
hypoxic areas (Dıaz 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 fluctuating 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 (Air-
ries and McMahon 1994; McMahon 2001). Below the
critical oxygen tension (Pc), however, increases in venti-
lation rate are unable to compensate for hypoxia, and
ventilation frequency decreases together with oxygen
uptake (Airries and McMahon 1994). In hypoxic environ-
ments, most crustacean species also respond by exhibiting
bradycardia, thus limiting the amount of energy expended
by the cardiovascular system. Furthermore, crustaceans can
alter blood flow during hypoxia, redirecting blood to tis-
sues 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 oxy-
gen 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 res-
piration follows the oxygen concentration (Vernberg 1983).
This point has been defined 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 redu-
ces 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 sufficient to understand how an environmental factor,
like dissolved oxygen (DO), modulates the physiological
adaptation of benthic crustaceans. In fact, the hypoxia-
inducible factor (HIF), conserved during evolution from
worms to flies 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 pro-
duced by low dissolved oxygen levels are generally
compensated by an increase in other physiological
responses, despite reduced respiratory efficiency. 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 modifications of the hemolymph acid–base
balance (Martinez et al. 1998), hemocyanin binding
capacity, oxyhemocyanin-protein relationship, hemo-
lymph 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
flow 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).
8 Mar Biol (2010) 157:7–18
123
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 propheno-
loxidase (proPO), which are involved in encapsulation,
melanization, and cytotoxicity, as a nonself recognition
system (Johansson et al. 2000). Environmental parame-
ters 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 flow diversion away from the hepatopancreas. A
reduction in protein synthesis was interpreted as a conse-
quence 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 shellfish fisheries 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 respon-
ses 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 oxyhem-
ocyanin: OxyHc), immune state (hemocytes concentra-
tion), digestive capacity (general protease, trypsin, and
chymotrypsin), and metabolic enzymes (lactate dehydro-
genase: 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 ± 0.5�C, 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 12�C and
31 psu. Twice a day, 1-lm UV filtered sea water was
exchanged at the corresponding oxygen concentration.
Nitrogen was used to reach the sea water’s 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) pie-
ces, 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 indi-
vidually in sea water (0.22-lm filtered UV-treated seawater
at 12�C and 31 psu) in 1,000-ml hermetic chambers at each
experimental DO level (closed respirometer). Oxygen
content was quantified before and after incubation (ca. 3 h)
by an optic sensor connected to a temperature-compen-
sated Microx MX3 AOT oxygen meter (PreSens, GmbH,
Germany) calibrated with saturated sea water (100%) and
5% sodium sulfite solution (0%). Three chambers without
animals were used per treatment as control.
Blood analysis
Hemolymph sampling
After oxygen consumption measurements, five crabs were
randomly sampled for OxyHc, hemocytes concentration,
and blood proteins. Thereafter, each animal was frozen at
-30�C and freeze dried for 48 h. Hemolymph was sampled
individually with a chilled syringe needle inserted at the
Mar Biol (2010) 157:7–18 9
123
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 (4�C)
plastic foil (Parafilm@) 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 dilu-
ted 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 coefficient 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 (4�C) antico-
agulant (450 mM NaCl, 10 mM KCl, 10 mM HEPES,
10 mM EDTA-Na2, pH 7.3, 850 mOsm kg-1) (Vargas-
Albores et al. 1993). Hemolymph plus anticoagulant was
centrifuged at 800g for 3 min at 4�C, 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
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 4�C 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 five volumes
of 95% ethanol. Assay tubes were placed in an oven at
37–40�C for 3 h. After precipitation, the assay tubes were
centrifuged at 3,340g for 15 min. The supernatant was
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 stan-
dard 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
4�C. 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. Diges-
tive gland homogenates were further diluted 1:500 for
protein determination by the Bradford technique (Brad-
ford 1976) adapted to a microplate method using a
commercial chromogen reagent (Sigma, cat. 610) and
bovine serum albumin as standard solution.
Digestive enzyme activity
General proteases activity was estimated in crude digestive
gland homogenates using azocoll (Sigma A4341) as sub-
strate in phosphate buffer, pH 7.5. Absorbance was mea-
sured in a spectrophotometer (SPECTRONIC model 21D)
at 520 nm. For this method, one unit was defined 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. Chy-
motrypsin enzyme activity was assayed in crude homoge-
nates 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 coefficient 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.
10 Mar Biol (2010) 157:7–18
123
Gill lactate dehydrogenase
Gills (8 ± 2.1 mg) were rehydrated using 200 ll pyrogen-
free 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 dehydroge-
nase, 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 4�C for 20 min, and 10 ll of
supernatant was added to 1 ml LDH reagent. LDH reagent
was preincubated at 37�C 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 coefficient of 6.22 for NADH.
Gill proteins were quantified similarly to digestive gland
proteins.
Statistical analysis
Normal distribution and homogeneity of variances were
tested with Kolmogorov–Smirnov and Levene median
tests, respectively. When the data did not satisfy the
prerequisites for parametric tests (analysis of variance,
ANOVA), Kruskal–Wallis H-tests were used to evaluate
the effect of oxygen tension on oxygen consumption,
oxyhemocyanin concentration, hemolymph protein con-
centration, hemocytes level, digestive gland metabolites,
and enzymatic activity. When the probability of error for
rejecting the null hypothesis was higher than 0.05, differ-
ences were considered not significant (NS). Otherwise,
a posteriori Holm–Sidak 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 fitted
to a linear regression with respect to the oxygen tension,
using the least-squares method. Other metabolites did not
satisfy the prerequisites for linear fitting. An R*C test of
independence was used to analyze effects of treatments on
mortality (Sokal and Rohlf 1995).
Results
No significant differences were detected in size and weight
of crabs from the five 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).
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 con-
sumption 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 nonsignificant
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
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
VO
2 (m
gO2 *
h-1*I
nd-1
)
b
c
a
Fig. 1 L. santolla. Individual oxygen consumption rate related to
dissolved oxygen level. Mean ± standard deviation. Significant
differences among treatments indicated by different letters (ANOVA,
P \ 0.05)
Mar Biol (2010) 157:7–18 11
123
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 fluctuated
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 significant 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 rela-
tionship between DO and these blood metabolites is
described by the following equations:
Glucose (mg gW�1Þ ¼ 5:836þ ð0:352 DOÞ;r2 ¼ 0:343; P ¼ 0:005
Cholesterol (mg gW�1Þ ¼ 2:657þ ð0:233 DOÞ;r2 ¼ 0:262; P ¼ 0:012
Acylglycerol (mg gW�1Þ ¼ 15:947þ ð2:335 DOÞ;r2 ¼ 0:221; P ¼ 0:021
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
Pro
tein
(m
g*m
l-1)
Prot
Oxy-Hc
a
b
c
c c
a
b
a
b b
Fig. 2 L. santolla. Hemolymph protein (Prot) and oxyhemocyanin
(Oxy-Hc) concentrations related to dissolved oxygen level.
Mean ± standard deviation. Significant differences between treat-
ments indicated by different letters (ANOVA, P \ 0.05)
0
500
1000
1500
2000
2500
3000
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
He
moc
ytes
(ce
ll*m
m-3
)
a
b
Fig. 3 L. santolla. Hemocytes concentration related to dissolved
oxygen level. Mean ± standard deviation. Significant differences
between treatments indicated by different letters (ANOVA, P \ 0.05)
0
5000
10000
15000
20000
25000
30000
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
Ac
tivity
(IU
*mg
prot
ein-1
)
a)
0
200
400
600
800
1000
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
Act
ivity
(IU
*mg
prot
ein-1
)
b)
0
5000
10000
15000
20000
25000
30000
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
Act
ivity
(IU
*mg
prot
ein-1
)
c)
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 significant differences
among treatments for the three variables (ANOVA, P [ 0.05)
12 Mar Biol (2010) 157:7–18
123
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). How-
ever, 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 envi-
ronments 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 first group of crustaceans, values below 4 kPa could
indicate specific 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 classified 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).
a)
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
mg*
g W
-1
Prot
Glyc
0
3
6
9
12
15
18
21
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
mg*
g W
-1
0
50
100
150
200
250
300
350
400
450
500
mg*
g W
-1
Gluc
Chol
AG
b)
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 coefficients see text
(P \ 0.05)
0
50
100
150
200
250
300
350
0 2 4 6 8 10 12 14 16 18 20 22
Oxygen tension (kPa)
Gill
LD
H A
ctiv
ity (I
U*m
g pr
otei
n -1)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Lact
ate
(mg*
gW-1
)
LDH
Lactate
Fig. 6 L. santolla. Lactate content in digestive gland and lactate
dehydrogenase LDH activity in gills related to dissolved oxygen
level. LDH left axis (filled circles); lactate, right axis (open circles).
Mean ? standard deviation
Mar Biol (2010) 157:7–18 13
123
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 sit-
uation, L. santolla was able to turn on mechanism to either
enhance the affinity of the actual OxyHc concentration or
increase the hemocyanin level. In the first case, the OxyHc
affinity 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 affinity 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 constitu-
ents) and eventually Mg concentration, affecting Hc
affinity. Another option is that hemocyanin synthesis acts
as a primary response to prevent cellular hypoxia.
Although, in the present study, a significant 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 first 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 up-
regulate genes of ATP synthesis-d- and ATP synthesis-f-
chains (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 first
response to low DO levels. These authors observed also
that after a 7-day exposure to chronic hypoxia, the adap-
tation induced by the 3-day exposure becomes insufficient,
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 specific
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 flow during hypoxia was not
measured, reduction in digestive gland metabolites (glu-
cose, 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: 2–4 kPa
(0.597 ± 0.709, 0.949 ± 1.019 mg W h-1 ind-1, respec-
tively) and 9–21 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, Holm–Sidak 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 pro-
cesses 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.
14 Mar Biol (2010) 157:7–18
123
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 concen-
tration 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 physio-
logical strategy to maintain shrimp during short fasting
periods. In another study, Muhlia-Almazan and Garcıa-
Carreno (2002) showed that total digestive proteases,
trypsin, and chymotrypsin of L. vannamei remained con-
stant 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 (60–90%) (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 organ-
isms, nutritional condition, and ecological condition,
among others. In the present study, OxyHc/protein pro-
portion in L. santolla control group showed values (30.6%;
21 kPa DO) lower than those reported for shrimp, sug-
gesting species-specific physiological adaptations related
with the ecology of this crab species. Preliminary obser-
vations 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 con-
centrations 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 flow 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-mac-
roglobulin, 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 quantification 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 crusta-
ceans, 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 glu-
coneogenesis 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 [glyco-
gen = 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,
Mar Biol (2010) 157:7–18 15
123
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 flow rates through the
supra-esophageal ganglion did not change during feeding,
suggesting that the supra-esophageal ganglion was priori-
tized independently from the DO level (McGaw 2005).
Such a prioritization could affect hemocytes synthesis.
Studies made in shrimps demonstrated that the hemato-
poietic 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 flow 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 findings
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 reflects an immune suppression that
might provide a critical window for opportunistic patho-
gens 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 con-
tributed to improve the manuscript. The experiments comply with the
current Chilean animal care and manipulation legislation.
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