Effect of attractant stimuli, starvation period and food availability on digestive enzymes in the redclaw crayfish Cherax quadricarinatus (Parastacidae)

Chemical stimuli in crayfish have been extensively studied, especially in the context of social interactions, but also to a lesser extent in relation to food recognition and the physiological response of digestive enzymes. This is particularly important in commercial species in order to optimize the food supplied. The first objective of this study was to determine whether incorporation of squid meal (SM) in food (base feed, BF) acts as an additional attractant for Cherax quadricarinatus and, if so, the concentration required for optimal effectiveness. Incorporation of SM was evaluated through individual and group behavioral tests. The second objective was to analyze the effect of food availability on behavior and level of digestive enzyme activity after short-term (48 h) and long-term (16 d) starvation periods. To assess the effect of either starvation period, 3 different treatments were conducted: no feed (control), available BF, and BF present but not available. Individual and group behavior showed no differences among treatments with different percentages of SM inclusion in BF. The time spent in chambers with different percentages of SM was similar in all treatments. Levels of amylase activity and soluble protein, as a function of food availability after a shortor long-term starvation period, were not altered. Digestive enzyme activity was not affected after 2 d of starvation in response to the treatment. However, change was observed in enzymatic profiles after juveniles were deprived of food for 16 d. The main responses were given by lipase, protease and trypsin activity. Based on previous studies and the present results, we propose a hypothesis for a possible regulation of the digestive and intracellular lipase activities depending on food availability.

ABSTRACT: Chemical stimuli in crayfish have been extensively studied, especially in the context of social interactions, but also to a lesser extent in relation to food recognition and the physiological response of digestive enzymes.This is particularly important in commercial species in order to optimize the food supplied.The first objective of this study was to determine whether incorporation of squid meal (SM) in food (base feed, BF) acts as an additional attractant for Cherax quadricarinatus and, if so, the concentration required for optimal effectiveness.Incorporation of SM was evaluated through individual and group behavioral tests.The second objective was to analyze the effect of food availability on behavior and level of digestive enzyme activity after short-term (48 h) and long-term (16 d) starvation periods.To assess the effect of either starvation period, 3 different treatments were conducted: no feed (control), available BF, and BF present but not available.Individual and group behavior showed no differences among treatments with different percentages of SM inclusion in BF.The time spent in chambers with different percentages of SM was similar in all treatments.Levels of amylase activity and soluble protein, as a function of food availability after a short-or long-term starvation period, were not altered.Digestive enzyme activity was not affected after 2 d of starvation in response to the treatment.However, change was observed in enzymatic profiles after juveniles were deprived of food for 16 d.The main responses were given by lipase, protease and trypsin activity.Based on previous studies and the present results, we propose a hypothesis for a possible regulation of the digestive and intracellular lipase activities depending on food availability.
Crustaceans exhibit relatively slow and intermittent feeding activity and this has an impact on food acquisition and processing.These behavioral characteristics affect the physical properties of feed pellets, such as water stability (hydrostability), and as a consequence, water quality (Saoud et al. 2012).Inasmuch as food is a significant expense in aquaculture production systems, the need to maximize food consumption and reduce wasted food is fundamental for economic success (Lee & Meyers 1996).
Considering the importance of chemical signals during the development of crustaceans, it might be assumed that the incorporation of attractants to food would allow individuals to find potential food in a shorter period of time, increasing the possibility of ingestion (Mendoza et al. 1997).It has been demonstrated that squid meal acts as a stimulant, increasing food consumption in Homarus gammarus (Mackie & Shelton 1972), Penaeus stylirostris and P. setiferus (Fenucci et al. 1980), P. monodon (Smith et al. 2005), and Litopenaeus vannamei (Nunes et al. 2006).Similarly, shrimp protein hydrolysates stimulate feed consumption in C. quadricarinatus (Arredondo-Figueroa et al. 2013).There are few studies regarding the use of chemoattractant substances incorporated into the diets of cultured freshwater decapod crustaceans (Arredondo-Figueroa et al. 2013) and their effect on feeding responses (Tierney & Atema 1988, Lee & Meyers 1996, Kreider & Watts 1998).
Under natural conditions where crayfish may feed ad libitum on foods appearing in various forms and compositions, differences in digestive processes are likely to occur (Kurmaly et al. 1990).Crustaceans alternate between periods of feeding and non-feeding during their development as a result of sequential molting (Vega-Villasante et al. 1999).Molting involves several stages with different feeding behaviors, including the cessation of external food intake from late premolt through early postmolt; therefore, energy needs can be met with different available external food sources or lipid reserves.Digestive enzymes are used as a physiological response to fasting (Cuzon et al. 1980, Jones & Obst 2000, Muhlia-Almazán & García-Carreño 2002, Rivera-Pérez & García-Carreño 2011, Calvo et al. 2013).Artificiallyinduced fasting and starvation may allow elucidation of the metabolic routes used in hierarchical order during molting and may initiate alternative biochemical and physiological adaptation mechanisms (Barclay et al. 1983, Comoglio et al. 2008).The midgut gland of crustaceans is the main organ for synthesis and secretion of digestive enzymes (including proteinase, lipase and carbohydrase), absorption and storage of nutrients (lipids and glycogen), which can be mobilized during the non-feeding periods (Icely & Nott 1992, Ong & Johnston 2006).The level of the digestive enzymes in decapod crustaceans does not remain constant during the molt cycles (van Wormhoudt 1974) as a result of both internal and external factors such as starvation and the availability, quantity and quality of food.In C. quadricarinatus, Loya-Javellana et al. (1995) demonstrated that crayfish are potentially capable of regulating their digestive processes according to food availability.
In the present study, we focused on factors affecting feeding in C. quadricarinatus.Our main hypothesis was that chemical signals from food affect digestive enzyme activity, and this response is modulated by food availability and starvation periods.Our first objective was to determine whether squid additives make food more attractive to crayfish and, if so, what concentration of additives elicits maximum food searching behavior.The second objective was to analyze the effect of food availability on digestive enzyme activity after short-and long-term starvation periods.This information may be useful to understand food searching behavior, and to determine the modulating effect of food presence on digestive physiology in order to design new diets and maximize food handling for the species.

Effect of squid attractant on juvenile ability to detect food
For the behavioral experiment, a 30 × 40 × 20 cm glass aquarium without water flow was designed (Fig. 1A) based on Jaime-Ceballos et al. (2007).The aquarium was divided into 3 similarly-sized, parallel chambers: the middle chamber was used for acclimation, and the right and left compartments were used as 'attractant chambers'.The aquarium was placed inside a white box to minimize disturbance to crayfish behavior.Food containers (4.5 × 4.5 × 6 cm, Fig. 1B) consisted of an acrylic box surrounded by nylon mesh (1 mm mesh pore).There was a net tube (1.5 × 4.5 cm, diam.× length) inside the container to prevent small particles of food from falling out when the acrylic structure was moved by the animals.
The ingredient tested as a food attractant was squid meal (SM, Illex argentinus), and its inclusion in BF was analyzed.The protein concentrate extraction of SM was performed by the Soxhlet method, with isopropyl alcohol as a solvent.The protein residue was then oven-dried at 80°C for 24 h according to The ability to detect food was evaluated under 2 experimental conditions: individually (April 2012) and in groups (April 2013).Twenty individual juvenile behaviors were observed in the glass aquarium per treatment, except for the reference positive control (N = 10) (weight: 1.35−3.25 g; N = 110) and group behavior was observed with 4 juveniles (weight: 1.21 to 3.75 g) per experiment with 5 replicates for each treatment (N = 60).The group behavior experiment was only performed for Treatments (1), ( 3) and ( 5) due to the results of individual behavior experiments.
Test specimens were acclimated to BF for 1 wk prior to the assays, and behavioral experiments were always performed between 09:00 and 13:00 h in the presence of artificial light, in order to avoid any effects of circadian rhythms (Sacristán et al. 2013).All crayfish were starved for 48 h prior to behavioral evaluation, and all were at intermolt, since it has been suggested that the level of responsiveness varies from stage to stage of the molt cycle (Harpaz et al. 1987).Only test specimens with complete sensory append ages (i.e.antennae and antennules) were selected.
At the beginning of each assay, juveniles were maintained in the acclimation chamber for 10 min as in Nunes et al. (2006).After each trial, water was discarded completely, the aquarium was washed with tap water and refilled with new filtered water.Water quality parameters were measured in order to avoid water quality effects on responses by test specimens to the chemoattractant (Lee & Meyers 1996).These parameters, i.e. dissolved oxygen (6 ± 1 mg l −1 ), pH (7.7 ± 0.5), hardness (80 ± 10 mg l −1 as CaCO 3 equivalents), and temperature (27 ± 1°C) were within the ranges recommended for aquaculture (Jones 1997, Boyd & Tucker 1998).
Behavioral response to the presence of the attractant was recorded visually by 1 observer positioned in front of the glass aquarium.The location of SM (i.e.left or right chamber) was chosen randomly for each behavior session.After acclimation, the glass doors of the chamber were opened and the following variables were evaluated: (1) first choice (SM or no SM) of the juveniles, and (2) residence time in each chamber for 10 min (a period established in a pre liminary bioassay).The food amount (BF, SM+BF or TetraColor) offered in each trial was 5% of the mean body weight of all crayfish.The percentage of positive choice was calculated as: positive choice (%) = (total number of positive choices / total number of comparisons) × 100, as in Nunes et al. (2006).The % residence time was calculated as: residence time (%) = (total time of positive residence / total assay time) × 100.

Effect of food availability on digestive enzyme activity
To evaluate the effect of food availability on digestive enzymes, 2 experiments were performed according to length of starvation period (short or long).In both experiments, treatments were: (1) no BF (control), (2) available BF (ABF), (3) BF present but not available (NABF).For each treatment, an 18 × 35 × 19 cm plastic aquarium was used; food was unprotected in the ABF treatment but was protected by a food container in the NABF treatment.Either the food or the food container was placed in the middle of the aquarium.In the ABF and NABF treatments, the amount of food offered was 5% of the juvenile's weight.
Expt 2: long-term starvation period A total of 72 intermolt phase crayfish (weight: 1.75 -5.17 g) were selected and starved for 16 d in individual plastic containers (500 cm 3 ) filled with 350 ml of de chlorinated water under continuous aeration.These containers were placed in 53 × 40 × 12 cm aquaria with water maintained at 27 ± 1°C.Starvation days were established in preliminary studies.During this period, the plastic containers were cleaned and water was renewed 3 times a week (during experiments no molting organisms were observed).Thereafter, the same procedure as in Expt 1 was performed, but the analysis times were 0, 30 and 120 min; at each time 8 crayfish were anesthetized in cold water and the midgut gland was dissected.

Enzymatic preparation and activity assays
At the end of short-and long-term starvation experiments, the midgut glands were dissected, weighed (± 0.1 mg) and immediately frozen at −80°C.Each midgut gland was homogenized in Tris-HCl buffer (50 mM, pH 7.5, 1:4 w/v) in an ice-water bath, with a Potter homogenizer.After centrifugation at 10 000 × g for 30 min at 4°C (Fernández Gimenez et al. 2009), the lipid layer fraction was removed and the supernatant was stored at −80°C until used as an enzyme extract for the enzymatic analysis.The absorbance of enzymatic assays was read on a JASCO CRT-400 spectrophotometer.
The amount of total soluble protein was evaluated with the Coomassie blue dye method according to Bradford (1976) using serum bovine albumin as the standard.Total proteinase activity was assayed using 1% azocasein as the substrate in 50 mM Tris-HCl, pH 7.5 (García-Carreño 1992).One proteinase unit was defined as the amount of enzyme required to increase 0.01 optical density (OD) units min −1 at 440 nm (López-López et al. 2005).Lipase activity of each enzyme extract was determined according to Versaw et al. (1989).The assay mixture consisted of 100 µl of sodium taurocholate 100 mM, 1900 µl of buffer Tris-HCl (50 mM, pH 7.5) and 20 µl of enzyme extract.After pre-incubation (25°C for 5 min), 20 µl of β-naphthyl caprylate substrate (Goldbio N-100) 200 mM in dimethyl sulfoxide (DMSO) was added.The mixture was incubated at 25°C for 30 min.Then 20 µl Fast Blue BB (100 mM in DMSO) was added.The reaction was stopped with 200 µl of tri chloroacetic acid (TCA, 0.72 N), and clarified with 2.76 ml of ethyl acetate:ethanol (1:1 v/v).Absorbance was recorded at 540 nm.One lipase unit was defined as the amount of enzyme required to increase 0.01 OD units min −1 at 540 nm (López-López et al. 2005).
Amylase activity of each extract was determined according to Vega-Villasante et al. (1993).The assay mixture consisted of 500 µl Tris-HCl (50 mM, pH 7.5) and 5 µl enzyme extract; 500 µl starch solution (1% in Tris-HCl, 50 mM, pH 7.5) was added to start the reaction.The mixture was incubated at room temperature for 10 min.Amylase activity was determined by measuring the production of reducing sugars resulting from starch hydrolysis as follows: immediately after incubation, 200 µl of sodium carbonate (2 N) and 1.5 ml DNS reagent were added to the reaction mixture and the mixture was boiled for 15 min in a water bath.The volume was adjusted to 10 ml with distilled water, and the colored solution was read at 550 nm.Reference tubes were prepared similarly, but crude extract was added after the DNS reagent.One amylase unit was defined as the amount of enzyme required to cause an increase of 0.01 OD units min −1 at 550 nm (López-López et al. 2005).
Trypsin activity was assayed according to Erlanger et al. (1961).The substrate solution was prepared using 100 mM benzoyl Arg-p-nitroanilide (BAPNA) dissolved in 1 ml of DMSO and brought to a volume of 100 ml with Tris-HCI 50 mM, pH 8.2 containing 10 mM CaCl 2 .Activity was measured by mixing 80 µl enzyme extract and 1.25 ml of substrate solution, and then the mixture was incubated for 20 min at 37°C.Subsequently, 0.25 ml of acetic acid was added, and the hydrolysis of BAPNA was determined by measurement of free p-nitroaniline at 410 nm.The trypsin activity was measured at 0, 30, and 120 min for Expts 1 and 2.

Statistical analysis
The positive choice and residence time data derived from paired comparisons of feeding behaviors were tested using the chi-squared test of independence (Zar 1999) and 1-way ANOVA (Sokal & Rohlf 1995) respectively.Digestive enzyme data from the short-and long-term starvation experiments were analyzed using generalized linear mixed models (GLMMs) with the statistical program R and the GLMMs package (Zuur et al. 2009), including treatments (control, ABF and NABF) and time as fixed factors.The significance level was set at α = 0.05.

Effect of chemoattractant on juvenile response
The results of individual and group crayfish behaviors are shown in Table 2.For individual crayfish response, no significant differences were found among treatments with different percentages of SM in cluded in the BF.Residence times in the chambers with different percentages of SM were similar in all treatments (p = 0.22).Group behavior showed that the percentage of positive choice was the same for all treatments (p > 0.05).Additionally, the crayfish did not preferentially stay in the chamber with the attractant (p = 0.91).

Expt 1: short-term starvation period
The results of specific enzyme activity for amylase, lipase, protease, trypsin and soluble protein level in the short-term starvation experiment are presented in Fig. 2. The digestive enzyme profiles and soluble protein from midgut gland extracts showed a similar pattern among treatments.Specifically, crayfish from the NABF treatment had significantly lower levels (p < 0.05) of amylase activity at 5 and 120 min (5.24 and 5.14 U mg protein −1 respectively) than the control and ABF (Fig. 2A).No significant difference was found between ABF and the control group (p > 0.05).Lipase activity of crayfish was not significantly affected (p = 0.19) by the treatments over the 120 min period of the experiment (Fig. 2B).Protease activity in the midgut gland of the juveniles in the NABF treatment was significantly lower (1.02U mg protein −1 ) than those in the control and ABF treatments at 5 min (p < 0.05) (Fig. 2C).Moreover, the crayfish in the ABF group differed significantly from the control (p < 0.05) only at 30 min.
Trypsin activity showed significant differences (p < 0.05) among control, ABF and NABF at 120 min (Fig. 2D); furthermore, the soluble protein level of the crayfish was not significantly affected (p = 0.47) by the treatments over 120 min (Fig. 2E).

Expt 2: long-term starvation period
The effect of food availability on the digestive enzyme activity of crayfish after long-term star vation is shown in Fig. 3.There was no significant difference in amylase activity (p = 0.37) among treatments over the 120 min observation period (Fig. 3A).Lipase activity of ABF exhibited a significantly lower activity (p < 0.05) than NABF at 30 and 120 min (61.52 and 48.31 U mg protein −1 respectively) (Fig. 3B).There were significant differences in protease activity (p < 0.05) among NABF, control and ABF at the initial time (Fig. 3C).At 30 min, the protease activity in NABF (1.15 U mg protein −1 min −1 ) and ABF (1.18 U mg protein −1 ) was significantly (p < 0.05) lower than the control group.The ABF decreased significantly (p < 0.05) to 0.70 U mg protein −1 at 120 min.Trypsin activity for ABF showed significant differences (p < 0.05) compared to the control and NABF treatments at the end of the experiment (Fig. 3D), but the levels of soluble protein concentration in the crayfish starved for 16 d did not show significant differences (p = 0.13) among treatments during the time assayed (Fig. 3E).Similar results were found when total en zyme activity (U mg midgut gland −1 ) was calculated (data not shown).

DISCUSSION
Although crayfish have polytrophic feeding habits (Saoud & Ghanawi 2013), this study showed that squid protein extract in the tested concentration range did not increase the attractiveness of feed to Cherax quadricarinatus.This result disagrees with other studies on Pleoticus muelleri, Homarus gammarus, Litopenaeus vannamei, Penaeus monodon, P. setiferus and P. stylirostris (Mackie & Shelton 1972 et al. 1980, Díaz et al. 1999, Smith et al. 2005, Nunes et al. 2006) even though the SM and the method for incorporating it into the experimental feed were the same across all studies.However, these other studies were performed on marine crustaceans (lobsters and shrimp), whereas this study is the first to test the effectiveness of SM as an attractant for a freshwater decapod.The experiment on food detection shows that crayfish wander around the aquarium regardless of where food is localized.Subsequently, when the animal is sufficiently close to or in contact with potential food, their chemoreceptors play a fundamental role in acceptance or rejection of potential food (Heinen 1980).Thus, given the feeding habits of C. quadricarinatus and our results, we propose the hypothesis that C. quadricarinatus mainly finds food due to the time they invest in environmental wandering.Given that our food searching behavior experiments were carried out in stagnant water, and that odor plumes are strongly affected by flow dynamics for detection of chemical signals (Weissburg 2011), crayfish food detection should also be studied using flowing water.Additionally, food searching behavior could be analyzed in a Y-maze apparatus to enhance the sensitivity of the experimental setup.Mackie (1973) determined that the squid-soluble extract is rich in proline, glycine, alanine and arginine.According to Heinen (1980), glycine and arginine act as attractants in decapod crustaceans.Tierney & Atema (1988) found that cellobiose and sucrose are important feeding stimulants for Orconectes rusticus; the amino acids glycine and glutamate elicited feeding movements in O. rusticus and O. virilis crayfish.Amino acids are abundant in the tissues of marine organisms and they probably guide predators and scavengers to food.Amino acids may likewise signal food availability to crayfish (Tierney & Atema 1988).Further research is needed to determine if the use of complex ingredients such as krill meal, or simple components like amino acids can act as signals for food detection in C. quadricarinatus.
Under natural conditions where crayfish may feed ad libitum on food of various forms and compositions, differences in digestive processes are likely to occur (Kurmaly et al. 1990).In the study on foregut evacuation, return of appetite and gastric fluid in C. quadricarinatus, Loya-Javellana et al. (1995) demonstrated that ingested food was evacuated linearly with time in crayfish fed daily, and in a somewhat curvilinear trend in those fed every 2 d.This would indicate that crayfish are potentially capable of regulating their digestive processes according to food availability (Loya-Javellana et al. 1995).
Our results on starvation period and food availability demonstrated that crayfish starved for 48 h had a higher digestive enzyme specific activity than crayfish starved for 16 d.Relative to the highest activity level of amylase, lipase, protease and trypsin recorded during the analysis period of both starvation treatments, we found 4.95, 3.70, 0.29 and 1.12 times, respectively, more activity in crayfish starved in the short-term experiment compared to crayfish starved for the long-term.However, in terms of total activity, the differences were smaller, with similar total protease and trypsin activity, and only 60% higher lipase and amylase activity.Therefore, we can conclude that digestive enzyme activity is not affected after 2 d of starvation and in response to treatment.However, different enzymatic profiles were observed in C. quadricarinatus juveniles deprived of food for 16 d.The main responses occurred in lipase, protease and trypsin activity, which were higher in control and NABF groups; however, this may have been due to the protein provided by the food (in the case of specific activity), or due to the additional weight provided by the ingested food to the tissue (midgut gland), in the case of total activity; in both cases decreasing the enzyme activity.
The levels of amylase activity and soluble protein as a function of food availability after short-or longterm starvation were not altered.Calvo et al. (2013), analyzing C. quadricarinatus juveniles (1 g), observed that starvation did not have an effect on amylase activity, but an accentuated tendency to decrease after 50 d of starvation and to increase after 40 d of re-feeding.Our results are opposed to those of Clifford & Brick (1983), who found that fasting Macrobrachium rosenbergii use the energy from the oxidation of carbohydrates.
Our research demonstrates that C. quadricarinatus juveniles respond differently to food availability after a long-term starvation period (16 d).These results agree with Calvo et al. (2013), who observed low levels of lipase activity after a 50 d starvation period, suggesting that lipase is not synthesized when food is not available.In the same species, Yudkovski et al. (2007) demonstrated that lipase transcripts decrease in the hepatopancreas during non-feeding stages.
Studying the effect of starvation on the expression of lipase transcripts in Litopenaeus vannamei, Rivera-Pérez & García-Carreño (2011) showed that 2 types of lipase exist: a digestive lipase and an intracellular lipase (lysosomal).The digestive lipase is only found in the digestive gland and is negatively regulated during fasting by the absence of food, whereas the intracellular lipase is expressed in various tissues (digestive gland, uropods, pleopods, digestive tube, gills, hemocytes, muscle and gonad), and is positively regulated during starvation, suggesting that it is responsible for lipid mobilization from lipid depots (energy reserves).
Based on these previous studies and our present results, we propose a possible regulation pathway for the digestive lipase activity, which is summarized in Fig. 4. We hypothesize that the detection of food promotes de novo synthesis of digestive lipase (ABF and NABF treatments).Subsequently, manipulation, ingestion, stomach food content and nutritive molecules in the digestive gland stimulate digestive lipase secretion into the digestive gland ducts and stomach, interacting with food and carrying out degradation in both sections of the digestive tract (ABF).Therefore, the digestive lipase present in the stomach, intestine and digestive gland is acting on the food and cannot be fully quantified when the lipase activity is measured solely in the digestive gland (Fig. 4A).This would agree with the fact that digestive lipase activity does not increase when food is available (ABF).Therefore, the presence of food inside the stomach, and subsequent products of stomach digestion in the digestive gland and intestine would stimulate further degradation of the food.However, detection of food inhibits the intracellular lipase synthesis pathway, and thus stored lipids are not used as an energy source.
When there is no food in the environment for a long period of time, the intracellular lipase de novo synthesis is likely to be stimulated, and as a consequence, lipid stores mobilized.The pathway of digestive lipase synthesis is inhibited and digestive lipase remains at basal levels (Fig. 4B).In the present study, this assumption is supported by our observation of the low level of digestive lipase activity recorded in the control group after 16 d of fasting.However, when food is present but not available (only possible under experimental conditions), digestive lipase is synthesized and stored inside digestive gland cells and is not secreted.This could be due to the fact that there is no handling stimuli, ingestion, food content in the stomach, and food stomach digestion products in the digestive gland.However, because the synthesis of intracellular lipase is likely to be inhibited in the short-term, there may or may not be a mechanism to counteract this experimental effect (Fig. 4C) i.e. when C. quadrica rinatus detects the presence of food, the intracellular lipase synthesis is not active to restrict mobilization of lipid reserves and the synthesis of extracellular digestive lipase is also stimulated, in spite of the presence or absence of food in the stomach and digestive gland.This assumption is supported by our observation of increased activity of digestive lipase in the NABF treatment of this study.
Protease and trypsin activities reflected a similar response to food availability after 16 d of fasting.This result supports the concept that trypsin (together with chymotrypsin) is one of the main proteases of the digestive gland in decapod crustaceans, and it is believed to be responsible for 40 to 60% of total protein digestion in penaeids (Galgani et al. 1984, Tsai et al. 1986).
Our results show that the presence of available food stimulates trypsin secretion at 120 min after long-term starvation but not after short-term starvation.It is possible that during short fasts, the levels of digestive capacity do not decrease significantly because of the history of previous food.The result of the differential response of trypsin secretion under different fasting periods is related to the findings of Muhlia-Almazán & García-Carreño (2002), who reported that trypsin activity from the hepatopancreas in L. vannamei was diminished in response to fasting.Cuzon et al. (1980) demonstrated that trypsin activity in shrimp decreases during starvation.In turn, C. quadricarinatus juveniles exposed to nonaccessible food after 16 d of fasting show an increase in trypsin activity relative to the ABF group, which would indicate that it might be stored inside digestive gland cells until food entry to the digestive system.This is also related to the results reported by Hernández-Cortés et al. (1999), who demonstrated the presence of trypsinogen in the digestive gland of the crayfish Pacifastacus leniusculus.Furthermore, Sainz et al. (2004), in their study on trypsin synthesis and storage as zymogen in fed and fasted individuals of L. vannamei, revealed that trypsinogen is not totally secreted from a single cell, but rather appears to be secreted partially as an effect of ingestion.It must be considered that trypsinogen can be spontaneously reactivated during the preparation of enzyme extracts and therefore be quantified as an active enzyme, such that it might not be distinguished from the enzyme that is activated and secreted for food digestion by natural causes (Sánchez-Paz et al. 2003).Therefore, more studies in C. quadricarinatus are needed to clarify this issue.More studies are also needed regarding possible changes in messenger expression, as well as immunohistochemical studies of digestive tract cells as a reflection of physiological changes in the digestive enzyme secretion in this species.We observed a differential response (in terms of reaction time) in lipase, protease and trypsin activity when food became available again after pro- Lipase responded rapidly (after only 30 min) to the presence of food, whereas protease and trypsin levels responded only after 120 min.Therefore, these enzymes could be used as a tool to analyze the nutritional status of C. quadricarinatus.
During periods of starvation, crustaceans must use their energy reserves to meet their needs, and so enzymatic activity must be finely regulated to degrade the necessary energy reserves while preserving cell integrity as much as possible (Sánchez-Paz et al. 2006).Hence, changes in food intake during development may have important consequences for life history (Brzek & Konarzewski 2001).Although instantaneous ecological consequences of poor and sporadic nutrition are sometimes difficult to identify, the reproductive potential of any organism experiencing such conditions may be reduced (Sánchez-Paz et al. 2006).
Acknowledgements.This study was part of H.J.S.'s postgraduate scholarships (ANPCYT and CONICET) and PhD dissertation (University of Buenos Aires, Argentina).We are grateful to Dr. Raymond Bauer, University of Louisiana, Lafayette, LA, USA, and to the reviewers for their comments to improve the manuscript, to Lic.Amir Dyzenchauz for language revision, to Centro Nacional de Desarrollo Acuícola (CENADAC, Argentina) for providing the reproductive stock and to Dr. Gerado Cueto for his help with the statistical analysis.L.S.L.G. is grateful to Agencia Nacional de Promoción Científica y Tecnológica (PICT 2007, project 01187 and 2012 project 01333), CONICET (PIP 2009-2011, number 129 and PIP 2012-2014) KEY WORDS: Chemical stimuli • Crustaceans • Digestive enzyme • Food searching behavior • Food attractants • Starvation