J OURNAL OF C RUSTACEAN B IOLOGY, 32(6), 940-948, 2012
ADAPTIVE DIFFERENCES IN DIGESTIVE ENZYME ACTIVITY IN THE CRAB
NEOHELICE GRANULATA IN RELATION TO SEX AND HABITAT
Juan Pablo Lancia ∗ , Analia Fernández Gimenez, Claudia Bas, and Eduardo Spivak
Departamento de Biología e Instituto de Investigaciones Marinas y Costeras (IIMyC), Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Mar del Plata, Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Casilla de Correo 1245, 7600, Mar del Plata, Argentina
ABSTRACT
Neohelice granulata (Dana, 1851) is a semi-terrestrial burrowing crab that inhabits estuaries and saltmarshes feeding on grasses or sediment
depending on the microhabitat they occupy (vegetated saltmarsh or bare mudflat). Specific cellulolytic, amylolytic, and proteolytic enzyme
activities were analyzed in midgut gland homogenates of males and females from each microhabitat fed in the laboratory with Spartina
densiflora leaves and sediment, respectively, in order to detect sex, food and microhabitat related differences. The presence of β-1,4glucosidase, endo-β-1,4-glucanase, α amylase, trypsin and chymotrypsin were confirmed. Specific cellulolytic activity was higher in crabs
fed on leaves than in those fed on sediment or in no fed controls and variable differences between sexes were observed. Specific amylase
activity of crabs fed on leaves was the lowest recorded. Trypsin and chymotrypsin specific activities were higher in saltmarsh crabs fed on
leaves than in mudflat crabs fed on sediment. Different mechanisms of enzyme regulation to explain the observed differences among groups
were suggested. Additionally, differences between sexes suggest different metabolic needs related to gonad maturation. It is concluded that
N. granulata has the ability to adapt digestive enzyme production to support its physiological and metabolic needs based on the different
food sources available at each microhabitat.
K EY W ORDS: digestive enzymes, feeding ecology, Neohelice granulata, nutrition
DOI: 10.1163/1937240X-00002090
I NTRODUCTION
The feeding pattern of crabs in the natural environment includes herbivorous, carnivorous, scavengers, deposit feeders, and seldom filter feeding species although most of them
have the ability to deal with a variety of diets and could be
considered “opportunistic omnivores” (Warner, 1977). Different food types, e.g., whole animals with different skeletal parts, animal carrion, macroalgae, siliceous microalgae,
vascular plants, etc., require different digestive processing
strategies (different mechanical processing, enzymes, time
of digestion) and the understanding of the digestive process
constitutes a mechanistic bridge between the physiological
processes that occur in the digestive tract, feeding and nutritional ecology (Karasov and Martínez del Rio, 2007).
Generally, digestive enzymes in crustaceans are synthesized in the midgut gland and are subsequently released
into the gastric chamber forming the gastric juice (Icely and
Nott, 1992). Furthermore, the profile of digestive enzymes
of decapod crustaceans varies according to their particular
feeding strategies (Figueiredo and Anderson, 2009). For example, carnivorous species, whose diets are predominantly
composed of protein, may produce high levels of proteolytic enzymes such as trypsin and chymotrypsin. Omnivorous species express proteinase and carbohydrase activities, whereas herbivorous show high activities of cellulase
and hemicellulase enzymes as they ingest large amounts of
∗ Corresponding
carbohydrates from plant cell walls (Johnston and Freeman,
2005; Linton et al., 2009).
Many terrestrial and semiterrestrial crabs are primarily
herbivorous or detritivorous, although they may act as no
specialized predators or eat carrion whenever possible (Wolcott and O’connor, 1992). The opportunistic feeding of these
crabs makes them suitable models to examine the adaptive
modulation hypothesis. On the basis of the assumption that
natural selection acts to maximize net energy gain, this hypothesis predicts that features of digestive physiology are
flexibly matched to the prevailing diet, and one might expect a positive relationship between levels (including quantity and structure) of carbohydrates, proteins, and lipids in
the natural diet and the presence or amount of gut enzymes
necessary for the digestion (Karasov and Diamond, 1988;
Karasov and Martínez del Rio, 2007). As a consequence, intraspecific differences in digestive enzymes could appear as
a result of variable nutrient availability, e.g., microhabitat
differences. In addition, since crabs differ in energy requirements between sexes (females may feed more than males
due to higher energy requirements from ovogenesis, Cannicci et al., 1996) intraspecific differences in digestive enzymes could also appear as a result of diverse individual
needs.
Neohelice granulata (Dana, 1851), is a semi-terrestrial
burrowing crab that inhabits estuaries, lagoons and marine
coasts of the southwestern Atlantic and that has received
author; e-mail: [email protected]
© The Crustacean Society, 2012. Published by Brill NV, Leiden
DOI:10.1163/1937240X-00002090
LANCIA ET AL.: DIGESTIVE ENZYME ACTIVITY IN N. GRANULATA
special attention in ecological, physiological and biochemical studies (Spivak, 2010). It is considered herbivorous and
detritivorous since it feeds on halophytic grasses (Spartina
spp.) in saltmarshes, but shifts to sediment rich in organic
matter in unvegetated tidal flats (Iribarne et al., 1997). The
organic matter found in sediment comes mainly from the
degradation of species of the cord-grass Spartina, and from
some protein-rich items such as small crustaceans, polychaetes, and nematodes (Botto et al., 2006). In the intertidal area N. granulata is exposed to a great number of
changing physical (temperature, humidity, salinity, and dissolved oxygen in water) and biological (food items) variables, all of which can produce changes affecting feeding
and metabolism (Oliveira et al., 2004). In addition, crabs
feed on sediment when tides recedes but on plants during
flooding tides (Bas, personal observation) and, consequently,
they face different sets of conditions while feeding at each
habitat.
Differences at the biochemical level (activity of amylase, disaccharidase, lipase, total proteolytic enzymes, and
reserves mobilization) have been observed in males of N.
granulata coming from adjacent saltmarsh and mudflat areas
and attributed to different food sources (Pinoni et al., 2011).
On the other hand, Bas et al. (unpublished) observed the lack
of feeding activity of N. granulata in the field during periods
of at least 48 hours under different conditions along the year,
and Vinagre and Da Silva (2002) found that this is a fastingadapted species that is able to maintain metabolic homeostasis after fasting periods as long as three weeks, which may
be natural for these crabs.
The aims of this study are twofold: 1) to detect sex
related differences in digestive enzymes present in the
digestive gland of N. granulata, and 2) to refine the previous
findings concerning microhabitat (saltmarsh vs. mudflat
crabs) differences in those enzymes. To achieve the first
goal, the specific activity of proteolytic, cellulolytic, and
amylolytic enzymes was compared between males and
females collected in winter, when males are in a resting post
reproductive period while females, that do not feed during
the summer ovigerous period, are developing ovaries and
preparing for the next reproductive season (Ituarte et al.,
2006; Bas et al., unpublished). To accomplish the second
goal, the specific activity of the same enzymes was measured
in crabs collected in the saltmarsh and the mudflat, fasted
during 5 days and then fed on S. densiflora and sediment
from the natural habitat, respectively, in order to be confident
of the quality of the food ingested.
M ATERIALS AND M ETHODS
Crab Collection and Food Preparation
The present study was carried out with crabs from Mar
Chiquita coastal lagoon (Buenos Aires province, Argentina.
37°35 S, 57°26 W), a brackish body water (46 km2 ) affected
by low amplitude (1 m) tides (Spivak et al., 1994). It is
characterized by a saltmarsh area dominated by halophyte
vegetation (Spartina densiflora and Sarcocornia perennis)
and a bare mudflat (Isacch et al., 2006). In order to maximize
homogeneity among specimens used in the experiments,
crabs were caught in winter, when they are in resting post
reproductive period and all females present the same ovar-
941
ian developmental stage (Ituarte et al., 2006). In addition, all
crabs were in intermolt stage (Drach and Tchernigovtzeff,
1967) and only a small adult size range was used. Twenty females (23.5 ± 2.8 mm of carapace width, CW) and 20 males
(26.5 ± 2.8 mm CW) were hand-collected from each habitat
(saltmarsh and mudflat) and immediately taken to the laboratory. All animals were kept five days without food to ensure
stomach emptiness before starting experiments; they were
placed in 50 l aquaria with 23h water salinity at 20°C.
The sediment used in the experiments was obtained from
the same mudflat where crabs were caught by scraping the
superficial layer of soil (5 mm thickness) that contained
algae and microorganisms deposited by water at high tide.
Green leaves of S. densiflora were randomly cut with
scissors from different plants from saltmarsh. All the food
was stored in bags and frozen (−20°C) to be defrosted only
when the feeding experiments were performed.
Animal Feeding and Midgut Gland Extraction
The midgut gland was used to evaluate the enzymatic
activity, instead of gastric juices in stomach, which offered
us the simplicity of extraction and the possibility to obtain
greater volumes.
After five days of fasting, 10 males and 10 females
collected from the saltmarsh were fed one hour (between
12 and 1 P.M.) with leaves and other 10 males and 10
females continued without food as a control group. In a
similar way, 10 males and 10 females collected from the
mudflat were fed with sediment one hour, at the same hour,
and a similar group was kept without food as control. This
feeding period is enough to ensure the fullness of crab’s
stomach (Lancia, personal observation). Both types of food
were offered differently: crabs fed with sediment (SM and
SF, males and females respectively) were placed separately
in aquaria (70 × 40 cm) without water, and wet sediment
was offered in plastic plates inside the aquaria; crabs fed
with leaves (LM and LF, males and females respectively)
were put into similar aquaria but full of 23h salinity water,
and S. densiflora leaves were offered inserted into a piece of
polypropylene attached to a stone.
All specimens (fed and not fed controls) were cryoanesthetized and rapidly killed by introducing forceps ventrally in the cephalotorax, immediately after the one-hour
feeding period expired. To extract the midgut gland, carapace was dorsally opened and the organs removed with forceps, placed in microtubes on ice and stored at −20°C.
Stomachs of fed individuals were checked for food and replaced if they were empty.
Biochemical Assays
Initially, specific enzyme activity was evaluated individually
on ten midgut glands of crabs of each treatment, in order
to determine the variability among them. Considering that
no significant statistical differences were found among enzymatic activity of individuals of the same treatment (t test,
all P values > 0.05) and the small size of individuals (and
the subsequent small volume of midgut gland obtained),
all midgut glands from the same experimental group were
pooled to ensure enough material for all analysis, homogenized with chilled distilled water (1:3 w/v) and centrifuged
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
(Presvac EPF-12R) for 30 min (10 000 g at 4°C) according to Fernández-Gimenez et al. (2002). Measurements of
all assays were made by triplicate. Soluble protein, used as
reference to express specific enzyme activity, was evaluated
in the supernatants (Bradford, 1976), bovine serum albumin
was used as standard (1 mg ml−1 ).
Optimum pH was determined for specific cellulase, β-1,4glucosidase, endo-β-1,4-glucanase, amylase and proteinase
activities before performing each activity assay (data not
shown).
Specific cellulase activity and those of β-1,4-glucosidase
(cellobiase; EC 3.2.1.21) and endo-β-1,4-glucanase (EC
3.2.1.4) were evaluated according to Linton and Greenaway
(2004). Reactions and incubations were carried out at 40°C
in 1.5 ml centrifuge microtubes in a thermal bath with agitation to allow comparisons with data for cellulase activities of other invertebrates. Reducing sugars were determined
with the dinitrosalicylic acid (DNS) method modified from
Miller (1959). Standard graph was obtained using different
concentrations (0 to 20 mg ml−1 ) of 1% glucose.
Specific cellulase activity was measured as the rate
of production of glucose from microcrystalline cellulose
(Sigmacell 20, Cat. No. S3504, Sigma Chemical Corp.,
St Louis, MO, USA). Homogenates (50 μl) were mixed
with 100 μl of 2% (w/v) microcrystalline cellulose made
up in 100 mM acetate buffer, pH 5.5. Suspension of
the cellulose was made by vortexing the stock cellulose
immediately before pipetting. A blank (50 μl homogenate +
100 μl buffer) was prepared for each sample analyzed to
enable correction due to the background absorption of the
homogenate at the wavelength measured. The buffer used
was 100 mM acetate, pH 5.5. The mixture was incubated
and agitated for 60 min at 40°C in a thermal bath (Vicking
Dubnoff SRL) before the reaction was stopped by the
addition of 25 μl of 0.3 M tri-chloro acetic acid (TCA). The
excess of acid was neutralized with 5 μl of 2.5 M K2 CO3
and the incubation mixture was centrifuged for 10 min at
10 000 g. Reducing sugar concentration was determined in
a 100 μl aliquot of the supernatant mixed with distilled
water and the DNS reagent [2 mM DNS in 0.2 N NaOH,
2.12 M Sodium Potassium Tartrate Tetrahydrate; VegaVillasante et al. (1993)] until complete 1 ml of solution,
the mixture was vortexed and placed in boiling water bath
for 15 min and cooled under running tap water to adjust
to room temperature. Distilled water (5 ml) was added
and the absorbance was read at 540 nm using an SP-2000
UV SPECTRUM spectrophotometer. The absorbance values
were then translated into glucose concentration using the
standard graph obtained before.
Specific β-1,4-glucosidase activity was measured as the
rate of production of glucose from cellobiose (Cat. No.
C-7252, Sigma Chemical Corp., St Louis, MO, USA).
Homogenate (25 μl) was mixed with 25 μl of 100 mM
acetate buffer (pH 5.5) and 50 μl of 14.61 mM cellobiose
in the same buffer, and the mixture was incubated at 40°C
for 30 min. The reaction was stopped by the addition of
25 μl of 0.3 M TCA and the excess of acid neutralized
with 5 μl of 2.5 M K2 CO3 . Precipitated protein was pelleted
by centrifugation for 10 min at 10 000 g. Blanks contained
25 μl of homogenate + 75 μl of buffer. Reducing sugars
concentration was determined in 100 μl aliquot of the
supernatant by the DNS method as described for specific
cellulase activity.
Specific endo-β-1,4-glucanase activity was measured as
the rate of production of reducing sugars from the substrate
carboxymethyl cellulose (Sigma Cat. No. C-5678, Sigma
Chemical Corp., St Louis, MO, USA). Homogenate (20 μl)
was mixed with 80 μl of buffer and 100 μl of 2% (w/v) carboxymethyl cellulose in the same buffer. Blanks contained
20 μl of homogenate and 180 μl of buffer. The buffer was
100 mM acetate, pH 5.5. Samples and blanks were incubated
at 40°C for 10 min and the reaction stopped by the addition
of 25 μl of 0.3 M HCl. The excess of acid was then neutralized by the addition of 5 μl of 2.5 M K2 CO3 and the incubation mixture was centrifuged for 10 min at 10 000 g. Reducing sugars produced were measured by the DNS method as
described before.
Specific amylase activity was measured following Bernfeld (1951) modified by Vega-Villasante et al. (1993) as the
rate of production of glucose from starch as substrate. Homogenate (10 μl) was mixed with 490 μl of 50 mM acetate
buffer (pH 5.5) and 500 μl of 1% starch (dissolved in buffer).
The mixture was incubated at 30°C for 20 min. Blanks contained 10 μl of homogenate and 990 μl of buffer. The incubation mixture was centrifuged for 10 min at 10 000 g and
the glucose produced during the incubation was measured
by the DNS method as described before. Enzyme activities
were expressed as μg of glucose min−1 mg−1 protein.
Specific proteolytic activity was assayed using 1% azocasein in 50 mM Tris-HCl, pH 7.5. Triplicates of 5 μl enzyme extracts were mixed with 0.5 ml of buffer and 0.5 ml of
substrate and incubated for 10 min at 25°C. Proteolysis was
stopped by adding 0.5 ml of 20% trichloroacetic acid (TCA),
mixture was centrifuged at 14 000 g for 5 min and supernatant absorbance was recorded at 366 nm (García-Carreño,
1992).
Specific trypsin and chymotrypsin activities could not be
evaluated in control crabs and only those from fed crabs
were obtained by measuring the rate of hydrolysis of synthetic substrates. Specific trypsin activity was measured
using N -benzoyl-DL-Arg-p-nitroanilide (BAPNA) as specific substrate. BAPNA (1 mM) was dissolved in 1 ml of
dimethylsulfoxide and adjusted to 100 ml with Tris-HCl,
pH 7.5 buffer, containing 20 mM CaCl2 . Triplicates of homogenates (5 μl) were added to 0.75 ml of substrate solution at 37°C and changes in absorbance at 410 nm were recorded at intervals of 30 sec. during 10 min (Erlanger et al.,
1961). Specific chymotrypsin activity was evaluated using
0.1 mM succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SAPNA)
in 0.1 M Tris-HCl, pH 7.5 containing 10 mM CaCl2 . Triplicates of homogenates (5 μl) were added to 0.75 ml of substrate solution at 37°C and changes in absorbance at 410 nm
were recorded at intervals of 20 sec. during 5 min (Del Mar
et al., 1979). Water and commercial enzymes (1 mg ml−1 )
were used as blanks and internal controls were carried out
respectively. Specific enzyme activities were expressed as
the change of absorbance min−1 mg−1 of protein of the enzyme used in the assays ( Abs min−1 mg−1 protein).
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LANCIA ET AL.: DIGESTIVE ENZYME ACTIVITY IN N. GRANULATA
Statistical Analysis
Differences in soluble protein, specific cellulase, β-1,4glucosidase, endo-β-1,4-glucanase, α amylase and proteinase activities were analyzed with three-way ANOVAs,
with food type (sediment, leaves), feeding condition (fed, no
fed) and sex as factors. Differences in specific activities were
evaluated with two way ANOVAs with food type and sex as
factors. Normality and homoscedasticity were checked and
data transformed when assumptions were not met. Pos-hoc
comparisons were performed with a Tukey test. In all cases,
significance was set as P < 0.05 (Sokal and Rohlf, 1979).
R ESULTS
Protein content of the midgut gland varied among different
experimental groups but in all cases was higher in control
than in fed animals (Table 1). Significant interactions were
present between pairs of factors (P < 0.001 in all cases).
When compared with control, crabs from the saltmarsh fed
with leaves showed a more noticeable decrease in protein
content than those from the mudflat fed with sediment.
Specific cellulolitic activity was detected in homogenates
of midgut gland from all groups, either with microcrystalline cellulose, cellobiose or carboxymethyl cellulose as
substrates. In general, the specific activity of cellulase,
β-1,4-glucosidase and endo-β-1,4-glucanase was higher in
crabs from saltmarsh fed on S. densiflora leaves than in
no fed controls and crabs fed on sediment (Figs. 1, 2
and 3, respectively). Males had higher specific cellulase and
β-1,4-glucosidase activity than females when fed on leaves
(Figs. 1b and 2b) and higher specific endo-β-1,4-glucanase
activity when fed on sediment (Fig. 3b), whereas control females from saltmarsh showed higher activity than males for
this last enzyme (Fig. 3a). The feeding condition was the
most influential factor on specific cellulase and endo-β-1,4glucanase activities (F = 97.86 and F = 55.77, respectively) but the type of food was the most influential factor
affecting specific activity of β-1,4-glucosidase (F = 67.38).
Specific amylase activity was higher in control crabs from
saltmarsh than in the remaining groups (Fig. 4). There were
no differences between control crabs from mudflat and those
fed on sediment. Amylase activity of crabs fed on leaves
was the lowest observed (Fig. 4b). The feeding condition
was the most influential factor on specific α amylase activity
(F = 73.39).
Specific proteinase activity was highly variable among
groups (Fig. 5a, b) and an interaction existed between type
of food and sex (P < 0.001). Activity was comparatively
high in males and females from the saltmarsh. Nevertheless,
Fig. 1. Specific cellulolytic activity in midgut gland homogenates of
Neohelice granulata. A, control (no fed) males (M) and females (F)
from saltmarsh and mudflat; B, males and females from saltmarsh and
mudflat fed on leaves and sediment respectively. Activity was expressed
as μg min−1 mg−1 protein of reducing sugars released. Mean of three
replicates + standard deviation. Significant differences are indicated by
different letters (Tukey test, P < 0.05).
when crabs of both groups were fed, specific activity
remained unchanged in males but decayed notoriously in
females (Fig. 5a, b). Control crabs from the mudflat showed
a lower specific activity and differences between sexes.
Furthermore, in this case, while fed males showed a lower
specific activity, fed females presented no different specific
proteolytic activity than controls. The feeding condition was
the more influential factor (F = 111.2).
Specific trypsin and chymotrypsin activities were higher
in saltmarsh crabs fed on leaves than in mudflat crabs fed on
sediment (Figs. 6 and 7). Females fed on sediment showed
higher trypsin activity than males (Fig. 6) whereas there
were no sex differences in chymotrypsin activity (Fig. 7).
Food type was the most influential factor on the specific
activity of both enzymes (F = 526.8 and F = 152,
respectively).
Table 1. Soluble protein content of Neohelice granulata midgut gland homogenates under different conditions. Mean of
the three replicates ± standard deviation. Significant differences are indicated by different letters (Tukey test, P < 0.05).
Protein content (mg ml−1 )
Starved
Saltmarsh
Mudflat
Fed
Male
Female
Male
Female
18.22 ±
20.88 ± 0.25b
18.25 ±
18.70 ± 0.42a
6.42 ±
14.73 ± 0.36d
8.58 ± 0.20e
13.47 ± 0.20d
0.19a
0.15a
0.71c
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
Fig. 2. Specific β-1,4-glucosidase activity in midgut gland homogenates
of Neohelice granulata. A, control (no fed) males (M) and females (F)
from saltmarsh and mudflat; B, males and females from saltmarsh and
mudflat fed on leaves and sediment respectively. Activity was expressed
as μg min−1 mg−1 protein of reducing sugars released. Mean of three
replicates + standard deviation. Significant differences are indicated by
different letters (Tukey test, P < 0.05).
D ISCUSSION
The specific activity of N. granulata digestive enzymes
showed differences between groups in most of the tested
substrates. This suggests different physiological conditions
of the individuals and supports the idea that this species
possess the potential capability to modulate their enzyme
activities depending on sex, habitat and food availability.
This plasticity may be considered as ecologically adaptive,
since this species inhabits environments where feeding is not
continuous (depending among other variables, on irregular
tidal cycles) and at the same time, the availability of one
or another type of food is influenced by the combination
of a series of physical and biological factors (Bas et al.,
unpublished). Although these crabs may move between
contiguous microhabitats, allowing an individual to shift
the food source between sediment and leaves of halophytic
plants (Luppi et al., 2012), they seem to prefer feeding on
the food available close to their burrows (Iribarne et al.,
1997) and a complex segregation of individuals has been
observed among microhabitats depending on sex and age
of crabs (Spivak et al., 1994). In addition, in winter (when
the experiments were performed), the species had finished
the reproductive period although females re-mature their
ovaries, even when reproduction is restored only at the end
of spring (Ituarte et al., 2004), while males are presumably
in a resting period. This could explain in some extent the
observed differences between sexes.
Fig. 3. Specific β-1,4-glucanase activity in midgut gland homogenates
of Neohelice granulata. A, control (no fed) males (M) and females (F)
from saltmarsh and mudflat; B, males and females from saltmarsh and
mudflat fed on leaves and sediment respectively. Activity was expressed
as μg min−1 mg−1 protein of reducing sugars released. Mean of three
replicates + standard deviation. Significant differences are indicated by
different letters (Tukey test, P < 0.05).
Much research has been done over diverse aspects of decapod crustacean digestive physiology, but the bulk of the
available studies deals with species of economic interest that
have a constant feeding activity and a high growth rate correlated with a protein rich diet (carnivorous). These studies were focused mainly on proteolytic enzymes (Naverrete
del Toro et al., 2006; Muhlia-Almazán et al., 2008), and
in a lesser extent, on carbohydrases. Based in those studies, the role of cellulolytic enzymes (and the role of vascular plants as food) were considered of minor importance
(Cuzon et al., 2000). However, a series of studies were performed more recently on cultured freshwater parastacids and
palaemonids, on mangrove crabs, and on terrestrial brachyurans (Pavasovic et al., 2004; Crawford, 2006; Linton and
Greenaway, 2007; Pavasovic, 2008). All these species rely,
in higher or lower proportion, on terrestrial plants or their
litter to sustain their growth and development, and present a
set of life history and metabolic adaptations to low nitrogen
and high fiber diets (Linton and Greenaway, 2007). These
studies clearly established the ability of some decapods to
use cellulose as energy source. Until not long ago (Martin, 1991) it was accepted that most of cellulolytic activity
detected in arthropods corresponded to the digestive skills
of protozoan and bacteria living in the intestinal tract of
their hosts, as occurs in the well-studied termites and terrestrial isopods. Within aquatic crustaceans, some isopods
seem to rely on bacterial endosymbionts to digest cellulose (Zimmer and Bartholmé, 2003). However, most crus-
LANCIA ET AL.: DIGESTIVE ENZYME ACTIVITY IN N. GRANULATA
Fig. 4. Specific α amylase activity in midgut gland homogenates of
Neohelice granulata. A, control (no fed) males (M) and females (F)
from saltmarsh and mudflat; B, males and females from saltmarsh and
mudflat fed on leaves and sediment respectively. Activity was expressed
as μg min−1 mg−1 protein of reducing sugars released. Mean of three
replicates + standard deviation. Significant differences are indicated by
different letters (Tukey test, P < 0.05).
taceans, including herbivorous, have very simple digestive
tracts and short time periods of food passage throughout the
gut (12 hours in omnivorous and herbivorous crabs from
marine shores and terrestrial habitats, respectively; Hopkin
and Nott, 1980; Greenaway and Linton, 1995) and therefore, have not enough time for bacterial fermentation that
might substantially contribute to the digestion of carbohydrates such as cellulose (Dall and Moriarty, 1983; Linton
and Greenaway, 2007). At the same time, direct molecular
evidence of endogenous endoglucanases has been recently
provided in a number of decapods (Crawford, 2006; Linton
et al., 2006; Allardyce and Linton, 2008) and in a terrestrial
isopod (Kostanjsek et al., 2010). Moreover, based on similar
evidence from different arthropods and nematodes, Watanabe and Tokuda (2001) suggested that endogenous cellulolytic enzymes may be a common trait in invertebrates.
The midgut gland extracts of N. granulata degraded cellulose in all its different forms since specific activity could
be detected from microcrystalline cellulose, carboxymethyl
cellulose and cellobiose as substrates. Nevertheless, the degree of endogenous or exogenous (coming from endosymbiont and/or free living microorganisms ingested with food)
enzyme activity cannot be defined from this study and
should be the subject of future research. Specific amylase activity was considerably higher than specific cellulolytic activity. This seems to be a distinctive trait in crustaceans since
945
Fig. 5. Specific proteinase activity in midgut gland homogenates of
Neohelice granulata. A, control (no fed) males (M) and females (F) from
saltmarsh and mudflat; B, males and females from saltmarsh and mudflat
fed on leaves and sediment respectively. Activity is expressed as the change
in absorbance min−1 mg−1 protein ( Abs min−1 mg−1 protein). Mean of
three replicates + standard deviation. Significant differences are indicated
by different letters (Tukey test, P < 0.05).
starch is considered the most efficiently degraded carbohydrate (Coccia et al., 2011).
In a similar way to amylase, specific proteolytic activity
and specific activities of both trypsin and chymotrypsin were
always present, which seem to be also a common trait among
many invertebrates, including decapod crustaceans (MuhliaAlmazán et al., 2008). Nevertheless, very different patterns
of activity among groups were observed when comparing
total and trypsin-like proteinase activities. Although trypsinlike enzymes are ubiquitous, other groups of proteinases,
as the aspartic group, have shown to be very important in
many decapod crustaceans (Naverrete del Toro et al., 2006),
having complementary activities, with pH optima different
Fig. 6. Specific trypsin activity (BAPNA as substrate) in Neohelice
granulata of fed (with leaves and sediment) males (M) and females (F)
crabs. Activity is expressed as the change in absorbance min−1 mg−1
protein ( Abs min−1 mg−1 protein). Mean of three replicates + standard
deviation.
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
Fig. 7. Specific chymotrypsin activity (SAPNA as substrate) in Neohelice
granulata of fed (with leaves and sediment) males (M) and females (F)
crabs. Activity is expressed as the change in absorbance min−1 mg−1
protein ( Abs min−1 mg−1 protein). Mean of three replicates + standard
deviation.
of those of serine proteinases (Le Boulay et al., 1996), or
even being the main group of proteinases present (Teschke
and Saborowski, 2005). Although no attempts were made
of measuring activities of other groups of proteinases,
one can hypothesize that the observed differences among
specific proteinase, trypsin and chymotrypsin activities in
the different experimental groups of N. granulata may be
a consequence of the differential activity of other proteinase
groups, e.g., aspartic proteinases (Le Boulay et al., 1996).
When all tested enzymes are considered, two types of
response seem to be possible when crabs are fed. In one
of them, observed in cellulases, the activity in the midgut
gland of crabs fed on leaves was higher than that in fasted
individuals (no response appeared when fed on sediment). In
the other, found in specific amylase and proteinase activities,
if a response existed, it consisted in a lower activity in fed
than in no fed control crabs. This must be related to the
way in which the activity of the corresponding enzymes
is regulated. During a digestive cycle, crustacean enzymes,
which are synthesized in the midgut gland, are released into
the cardiac chamber of stomach where food degradation
begins (Icely and Nott, 1992). Then, a drop of enzyme
activity in the midgut gland of crabs fed during one hour, as
it occurred with amylase and proteinases, suggest that those
enzymes were ready to be used, and were released early
in the digestive cycle. In contrast, a raised enzyme activity
in midgut gland of crabs after one hour of feeding (when
compared with fasted individuals), as observed in specific
cellulases activity, seems to indicate that the presence of
some type of food (leaves) in the stomach stimulates some
step in the synthesis or activation of enzymes, which are not
ready to act immediately after food ingestion. The complete
digestive cycle, and the enzyme activity in foregut and
midgut gland should be studied to test both hypotheses.
In general, enzyme activities were higher in crabs from
the saltmarsh fed with S. densiflora leaves than in crabs from
the mudflat fed with sediment. This could be related with
the need of degrade a low quality (protein poor: 0.7 to 5%
in dry weight, Mattson, 1980) and low digestibility (high
fiber content: up to 40% of dry weight constituted only by
cellulose, Karasov and Martínez del Río, 2007) vegetable
food source. A similar pattern of high proteinase activity was
observed in shrimps fed with low protein content food and
in herbivorous crabs (Johnston and Freeman, 2005). Some
animals feeding on plants respond in a compensatory fashion
by producing more enzymes (Karasov and Martínez del Rio,
2007), providing an ecological example of digestive enzyme
flexibility.
Sediment is supposed to be a better quality food source
taking into account that, in addition to plants debris (Botto
et al., 2005), some protein rich items as nematodes, polychaetes and small crustacean, are present (D’Incao et al.,
1990; Bas et al., unpublished). Nevertheless, no differences
were observed in some cases when comparing crabs fed on
sediment with unfed individuals. Feeding crabs with sediment in laboratory could have not closely resembled the natural feeding conditions, since sediment manipulation probably causes a mixing of the superficial layer of soil offered,
and a consequent decrease in the vegetable matter and small
organisms available for crabs.
It has been suggested by Pinoni et al. (2011) that the observed differences in total proteolytic activity and other biochemical traits between adult male N. granulata from mudflat and saltmarsh of the Mar Chiquita coastal lagoon, are
adaptive metabolic strategies to face the habitat variations.
The results of this study showed that differences in the specific cellulolytic, amylolytic, and proteolytic activities exist,
not only between food types but also between sexes. The observed plasticity is a promising starting point to understand
the ability of intertidal crabs to cope with discontinuous and
variable food sources in order to maximize the obtaining of
energy from the available food to fulfil the diverse requirements along their life cycles. In addition, it helps to understand the successful establishment and maintenance of dense
populations of this species in different habitats along its wide
latitudinal geographical distribution.
ACKNOWLEDGEMENTS
The authors would like to thank reviewers who greatly improved this
manuscript. This work is part of the doctoral thesis of J.P.L. and was
supported by a Ph.D. fellowship from National Research Council of
Argentina (CONICET-PIP 176) and Universidad Nacional de Mar del Plata
(EXA 527/10).
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R ECEIVED: 17 April 2012.
ACCEPTED: 11 June 2012.