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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