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Influence of feeding on hepatopancreas structure and digestive enzyme activities in Penaeus monodon.

ABSTRACT This study examines the influence of feeding on digestive enzyme activities in the black tiger prawn, Penaeus monodon, using histological and biochemical techniques. Juvenile P. monodon (>50 mm total length) were sacrificed after feeding at sequential time intervals, with unfed controls also sacrificed at the same time intervals. Resin histology revealed that there were no morphological changes in the digestive gland F-cells between fed and unfed P. monodon over time. There were no significant changes in [alpha]-amylase specific activities in fed animals over time post feeding, nor were there any changes in starved animals through time. Trypsin and [alpha]-glucosidase activities were significantly higher in unfed animals than fed animals. There was a significant peak in [alpha]-glucosidase activities at 0 min post feeding but this could not be conclusively determined as an influence from feeding because of high variability and the near negligible levels of [alpha]-glucosidase activities. A peak in lipase activity was observed at 60 min post feeding, but because there was no significant differences in lipase activities between fed and unfed animals it was therefore inconclusive. Results from histological and biochemical analyses failed to prove that feeding had a significant influence on digestive enzyme production of P. monodon and suggest that digestive enzyme production in P. monodon may be continuous and is not strongly influenced by feeding.

KEY WORDS: shrimp, digestive enzymes, F-cells, feeding, hepatopancreas, lumen, Penaeus monodon

INTRODUCTION

The most widely cultured prawn within the commercial prawn farming industry is the black tiger prawn, Penaeus monodon, because it has rapid growth rates and is larger than other farmed prawns at harvest. On such farms, food is often the highest operational cost (Chanratchakool et al. 1998). To be cost effective, prawn farms must achieve and maintain high feeding efficiencies while lowering food wastage. These requirements have paved the way for research into feeding strategies and penaeid enzyme studies, amidst other husbandry and nutrition studies.

Knowledge of penaeid enzyme dynamics, which is biologically important for the culture of these animals, has only been provided by generalist observations between feeding and/or feeding behavior in relation to digestive and cellular processes, or by direct histological examination. A continuous digestive enzymatic activity allows maximal digestive efficiency at all times but incurs a higher energy cost. Conversely, an induced response of increased enzyme secretion after food ingestion allows the organism to put more energy into somatic and/or gonadal growth because energy is not largely spent on continuous enzyme production. By clarifying the physiological aspects of feeding on the digestive functions, improved management of the cultured animals may be achieved.

Digestion in penaeids is facilitated by the hepatopancreas, which has several key roles including secretion of digestive enzymes, digestion and absorption of nutrients, storage of reserves and disposal of waste products (Al-Mohanna et al. 1985a, Al-Mohanna et al 1985b, Al-Mohanna & Nott 1987, Vogt 1993). It is bilobed and comprises numerous blind-ending tubules (Gibson & Barker 1979, Dall 1992, Brunet et al. 1994, Vogt 1994). These tubules are lined by epithelial cells, which are differentiated into 4 distinct types: E- (embryonic), F- (fibrillar), B- (blister-like) and R- (resorptive) cells (Jacobs, 1928). F-cells are involved in enzyme secretion. Al-Mohanna et al. (1985a), observed vesicles budding from Golgi bodies in the F-cells of fed green tiger prawn Penaeus semisulcatus and suggested that these vesicles were enzyme precursors or zymogens, but this is yet to be confirmed or observed in other penaeids (Dall 1992, Icely & Nott 1992). Vesicles were also observed in F-cells of the crayfish Astacus astacus after artificial draining of gastric juice (Vogt et al. 1989, Vogt 1996). The presence of digestive enzymes within these vesicles has been demonstrated by several authors with the use of immunohistochemistry (Malcoste et al. 1983), immunoflorescence (Vogt et al. 1989) and in situ hybridization (Van Wormhoudt et al. 1995).

The direct influence of feeding on enzyme production has been demonstrated in the European lobster Homarus gammarus by Barker and Gibson (1977). Using histology, they observed the discharge of supranuclear contents (digestive enzymes) from secretory cells after ingestion. Barker and Gibson (1978) found a similar trend from an identical experiment on the mud crab Scylla serrata. Similar results were also observed by Al-Mohanna et al. (1985a) in P. Semisulcatus, with the appearance of numerous zymogen granules in the F-cells corresponding to a maximal rate of enzyme discharge into the tubule lumen.

This study aims to clarify the effects of feeding on digestive enzyme production on the commercially important prawn P. monodon using histological and biochemical methods. By applying both techniques, confirmation of the effects of feeding on the hepatopancreas will hopefully be possible. The specific aims of this study are to examine the dynamics of the digestive gland in response to a single feeding event, by documenting changes in the structure of the hepatopancreas and the morphology of the F-cells and the specific activity of digestive enzymes. This was facilitated using a time series post feeding analysis on P. monodon juveniles.

MATERIALS AND METHODS

Animals and Husbandry

P. monodon post larvae were sourced from Rocky Point Prawn Farm, Queensland, Australia. The animals were housed in three 800-L tanks on a recirculation system and were fed daily to satiation with Artemia and commercial prawn pellets (Grobest t4, Primo Aquaculture).

Experimental Design and Procedure

The animals used in the experiment (TL > 5 cm) were randomly caught from the stock population using dipnets. Molt stage was determined by examination of setogenesis via changes in the seta on the margin of the inner uropod, in accordance with Smith and Dall (1985). Intermolt animals were randomly stocked into individual 1L containers to ensure that animals had equal access to feed and to prevent feeding hierarchies and cannibalism among conspecifics (Heinen 1987). The containers were then randomly allocated in the three holding tanks and kept afloat with Styrofoam sheets. The animals were placed under constant light to ensure that any enzymatic response was a function of feeding and not influenced by diurnal cycles. They were starved for 48 h to allow the animals to acclimatize to the containers as well as to ensure that stomach contents were emptied and that there would not be any residual effect from prior feeding regimes that would influence the results.

Fed animals were allowed to feed on commercial prawn pellets (Grobest T4, Primo Aquaculture) for 20 min. This feeding event was timed from the moment the animals collected the pellets with their periopods. At the end of 20 min, uneaten food was removed with a siphon. Unfed animals were disturbed in a similar fashion and were used as a control. At each of the times outlined in Table 1, five fed and five unfed animals were removed from their containers and placed in an ice slurry for 10 min to induce a chill coma.

The prawns were weighed and the hepatopancreas removed. Half the hepatopancreas was snap frozen in liquid Nitrogen and stored at -80[degrees]C for later digestive enzyme analysis. The remaining tissue was used for histology.

Wax Histology

One quarter of each hepatopancreas was fixed in Bouin fixative and processed routinely for wax histology. Sections (5 [micro]m) were cut using a Microm HM 340 microtome and stained with Hematoxylin and Eosin for structural examination or with Mercuric Bromophenol Blue to elucidate the location of proteins (Chapman 1975). Sections were examined at x100 magnification with an Olympus BH-2 light microscope. Images were scanned with a microscope-mounted Leica DC 300F digital camera and examined on an IBM computer using an image manager, Leica IM50. To determine if tubule lumen size changed in response to feeding over time, five tubules per slide were randomly selected using a numbered grid and a random number table. The area of the lumen and the tubule were measured using the freehand function of the image manager program.

Resin Histology

Approximately 2 [mm.sup.3] of hepatopancreas tissue from each animal was fixed in 5% gluteraldehyde in 2% sucrose-phosphate buffer pH 7.4 for 2-3 h, washed with 2% sucrose-phosphate buffer, dehydrated with ethanol and embedded in JB4 resin. Sections (2 [micro]m) were then cut and stained with polychrome stain. Changes in cellular structure in response to feeding and elapsed time after feeding/disturbance were described with emphasis on F-cell morphology because they are the enzyme secreting cells (Al-Mohanna et al. 1985a, Vogt et al. 1989, Vogt 1996).

Enzyme Extraction

Hepatopancreas tissue was thawed on ice and homogenized using an Ultra Turrax electric homogenizer (IKA-Werke, Germany) in 1 mL of chilled 0.1 M Tris 0.02M NaCl pH 7.0 buffer for 1 min. The homogenate was centrifuged at 10,000 rpm for 5 min to pellet debris and solids and the supernatant (subsequently referred as enzyme extract) stored at -20[degrees]C.

Enzyme Assays

One enzyme unit was defined as the amount of enzyme that catalyzed the release of 1 [micro]mole of product per minute and was calculated using the appropriate molar extinction coefficient ([member of]) in the assay conditions or a standard curve. Specific activity was defined as enzyme activity per mg of protein (Units mg [protein.sup.-1]). Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Spectrophotometric enzyme assays (200 [micro]L microassays) were performed in duplicate at 37[degrees]C in IWAKI flatbottom microplates and absorbances read in a Tecan Spectro Rainbow Thermo microplate reader. Appropriate controls were included with each analysis. Tests confirmed that enzyme activities were linear with incubation time.

Trypsin

Trypsin activity was measured using N-[alpha]-benzoylarginine-[rho]-nitroanalide (BAPnA) (Sigma B-4875) as the substrate. Each 200-[micro]L assay contained 180-[micro]L 0.2 M Tris 0.2 M NaCl pH 7.5 buffer, 10 [micro]L BAPnA dissolved in dimethylformamide (DMF) and 10 [micro]L enzyme extract. Trypsin activity was determined by measuring the release of p-nitroanalide at [A.sub.405nm] and using the molar absorption coefficient for [rho]-nitroanalide, 9,300 [m.sup.-1] [cm.sup.-1] (Stone et al. 1991).

[alpha]-Amylase

[alpha]-Amylase activity was measured using Ethylidene-[rho]NP-G7 (E-[rho]NP-G7) as the substrate. Each 200 [micro]L assay contained 195 [micro]L of Infinity amylase reagent (Sigma 568-20) and 5 [micro]L enzyme extract (thawed on ice) at 37[degrees]C. [alpha]-Amylase activity was determined by measuring the release of [rho]-nitrophenol at [A.sub.405nm] and using the molar absorption coefficient for [rho]-nitrophenol, 10 130 [m.sup.-1] [cm.sup.-1].

[alpha]-Glucosidase

[alpha]-Glucosidase activity was measured using [rho]-nitrophenol [alpha]-D-glucopyranoside (Sigma N-1377) as the substrate. Each assay, performed in a 37[degrees]C water bath, contained 350 [micro]L 0.1 M Tris 0.02 M NaCl pH 7 buffer, 100 [micro]L 0.02 M [rho]-nitrophenol [alpha]-D-glucopyranoside and 50 [micro]L enzyme extract. Aliquots (20 [micro]L) of the assay mixture were removed at 1 min and 40 min after addition of the enzyme extract and added to 180 [micro]L 1 M [Na.sub.2]C[O.sub.3] to stop the enzyme reaction, [alpha]-Glucosidase activity was determined by release of p-nitrophenol at [A.sub.400nm] and using the molar absorption coefficient for [rho]-nitrophenol, 18 300 [m.sup.-1] [cm.sup.-1] (Erlanger et al. 1961).

Lipase

Lipase activity was measured using 4-[rho]-nitrophenol caproate (4NPC) (SigmaN-0502) as the substrate. Each 200 [micro]L assay contained 190 [micro]L 0.5M Tris 0.1 M NaCl pH 8.5 buffer, 10 [micro]L of 2.5 mM 4NPC in ethanol and 10-[micro]L enzyme extract (thawed on ice) at 37[degrees]C. Lipase activity was determined by measuring by the release of [rho]-nitrophenol at [A.sub.405nm] and using the molar absorption coefficient for [rho]-nitrophenol, 19 800 [m.sup.-1] [cm.sup.-1] (Gjellesvik et al. 1992). A blank assay containing only 195 [micro]L buffer and 5 [micro]L substrate was also performed for each sample to deduct background absorbance from actual enzyme activity measurements.

Statistical Analysis

Chi-square tests of independence were used to determine if the area of tubule lumen changed as a function of feeding over time. The occurrence of autolysis in some animals resulted in insufficient replicates for statistical comparison (n < 3) of hepatopancreas structure from the sampling times of 120 min and 480 min post feeding. As a result, only the remaining 4 sampling times (0 min, 30 min, 60 min and 480 min post feeding) were analyzed from the histology. Orthogonal analyses of variance (ANOVA) were used to determine the effect of feeding on production of digestive enzymes. Homogeneity of variances was assessed using residual plots, and data were transformed with a square root transformation when the assumption of homogeneity of variances was violated.

RESULTS

Structure of the Hepatopancreas

The penaeid hepatopancreas consisted of numerous blind-ending tubules, each made up of a number of epithelial cell types. The epithelial cells rest on a basement membrane and surround a central lumen (Fig. 1A). F-cells stained positively in a darker blue with Mercuric Bromophenol Blue caused by the presence of proteins (Fig. 1B). The other cell types stained light blue except for the nucleus, where nuclear material was dark blue (Fig. 1B). Greater structural detail revealed from resin histology showed F-cells also had pink patches within the cytoplasm but their composition could not be determined from the polychrome stain (Fig. 1C).

[FIGURE 1 OMITTED]

There were no apparent differences in the morphology of the F-cells between the fed and unfed animals, nor were there any structural changes over time post feeding. There were also no zymogen granules or digestive vesicles observed.

The area of lumen did not change significantly over time post feeding (F = 2.703, df 3, 23, P = 0.069). It was also not significantly different between fed and unfed animals (F = 2.448, df 1, 23, P = 0.131). The mean lumen area was 37.8 [+ or -] 6.7% of the tubule area.

Digestive Enzymes

All of the enzymes tested were present in the hepatopancreas of the fed and unfed animals (Table 2), although activities were variable between individuals. The orthogonal ANOVA revealed that the interaction between enzyme dynamics, effect of feeding and elapsed time post feeding was not significant (Table 3). Therefore fed and unfed animals were grouped together when examining enzyme dynamics over time, and vice versa.

Trypsin

Trypsin specific activity in the unfed animals was twice as high as that of fed animals (Table 2, 3). There were no significant differences in trypsin specific activity over time post feeding/ disturbance (Table 3).

[alpha]-Amylase

There were no significant differences in o-amylase specific activity between fed and unfed animals (Table 2). In addition, o-amylase specific activity did not change significantly over time post feeding/disturbance (Table 3).

[alpha]-Glucosidase

Although [alpha]-glucosidase specific activities in both fed and unfed animals were low, the specific activity of [alpha]-glucosidase in the unfed animals was 3-fold higher than that of fed animals (Table 2). [alpha]-Glucosidase specific activity also significantly differed over time (Table 2). Specific activity at 0 min was approximately four times higher than the activity at 240 min post feeding/disturbance (Fig. 2).

[FIGURE 2 OMITTED]

Lipase

There was no difference in lipase specific activity between fed and unfed animals (Table 2). However, lipase activity differed significantly over time (Table 2). Lipase activity remained relatively constant at 0 min and 30 min then peaked at 60 min after which the activity declined significantly through to 480 min post feeding/disturbance. Lipase activity at 60 min post feeding was twice the activity than at 480 min post feeding/disturbance (Fig. 3).

[FIGURE 3 OMITTED]

DISCUSSION

This study is the first to examine the effect of feeding on digestive enzyme production in the commercially important black tiger prawn P. monodon using the combination of sensitive techniques, resin histology and spectrophotometric biochemical assays. These methods yield quantitatively more robust results than enzyme histochemical visualization used by Barker and Gibson (1977), histological analysis on F-cell differentiation after feeding via chemical tracers used by Al-Mohanna et al. (1985a) and immunohistochemistry with radiochemical tracers by Vogt et al. (1989). Most of previous research on digestive enzyme activities, as listed in Tables 4 and 5, were not used for analyzing the effect of feeding on digestive enzyme production, but were performed on animals that were fed constantly prior to analysis.

Furthermore, the short 20 min feeding period in this experiment was used to initiate a feeding response and associated digestive enzyme production and to allow for sampling at 30 min post feeding/disturbance (Table 1). A longer feeding period or the complete filling of the animals' gut was not necessary for the commencement of enzyme production. The 48-h starvation period for the animals was also implemented to ensure an empty gut prior to commencement of the experiment. This starvation period was sufficient and similar to the duration used by Al-Mohanna et al. (1985a) on P. semisulcatus. A week-long starvation period used on A, astacus by Vogt et al. (1989) was considered too long for a smaller tropical decapod like P. monodon.

The histological and biochemical results from this study suggest that P. monodon has a continuous digestive enzyme production, regardless of feeding, and contradicts previous studies that suggested an influence of feeding on enzyme production.

Effect of Feeding on F-cell Structure

The positive reaction of F-cells to Mercuric Bromophenol Blue confirms their role in digestive enzyme production. The negative reaction by B- and R-cells to Mercuric Bromophenol Blue also confirms that these cells do not function in enzyme secretion. These findings are consistent with several other studies (Dall & Moriarty 1983, Al-Mohanna et al. 1985a). Resin histology revealed pink patches within the cytoplasm of F-cells in both fed and unfed animals, but the nature of the organelles could not be conclusively determined from the polychrome stain (Fig. 1C). However, it is likely that these pink patches were rough endoplasmic reticulum (rER) or Golgi bodies, based on comparisons with previous studies on F-cell structure (Dali & Moriarty 1983; Al-Mohanna et al. 1985a). These structures could be better identified with electron microscopy.

Examination of fed and unfed animals post feeding in this study revealed a lack of structural differences in F-cells of both fed and unfed animals post feeding/disturbance. There was no evidence to suggest that F-cells of P. monodon had a distinct response to feeding through either digestive enzyme production via formation of zymogen granules (Al-Mohanna et al. 1985a) or appearance of vesicles (Vogt et al. 1989). This differs from several studies where histology demonstrated the influence of feeding on enzyme production.

Barker and Gibson (1977, 1978) demonstrated a positive response of enzyme production post feeding in the F-cells of the European lobster Homarus gammarus and the mudcrab Scylla Serrata within an hour after feeding. They observed three bursts of digestive enzyme discharge over a period of 5 h post feeding from the F-cells in both species, with each burst of activity measured by visual assessment of the intensity of the colored reaction product from histochemical sections. Al-Mohanna et al. (1985a) also reported a positive response of enzyme production post feeding in the F-cells of P. semisulcatas using electron microscopy, with one occurrence of enzyme production in the form of zymogen granules. However, it should be noted that no other authors have found zymogen granules in penaeids (Dall 1992, Icely & Nott 1992). Vogt et al. (1989) suggested that feeding had an influence on enzyme production, because enzyme vacuoles were observed in F-cells in fed crayfish Astacus astacus. The size of zymogen granules reported by Al-Mohanna et al. (1985a) were approximately that of Golgi bodies in F-cells and enzyme vesicles observed by Vogt et al. (1989) were similar in size to adjacent mitochondria. Although electron microscopy was not performed in the current study, F-cell organelles like Golgi bodies and mitochondria could be distinguished in resin sections. However, there was no evidence of zymogen granules or enzyme vesicles in the F-cells of P. monodon.

Furthermore, although these histological studies suggest that feeding influenced enzyme secretion, there is ambiguity in their conclusions because no starved controls were used. Thus the authors did not definitively demonstrate the influence of feeding on enzyme production, therefore it is possible that disturbance caused by feeding may be contributing to the observed changes and needs to be confirmed with the use of starved controls.

Analysis of Lumen Area After Feeding

During digestion, digestive enzyme secretions flow from the F-cells of the digestive gland into the tubule lumen and ultimately into the primary duct and then the foregut. Chyme (digestive fluid from the foregut) also migrates from the foregut to the hepatopancreas via the same route. It has been suggested that as the prawns feed, the hepatopancreas tubules become loaded with chyme and fine particles masticated by the gastric mill (Al-Mohanna & Nott 1987). However, there have not been any studies to elucidate changes in the size of the tubule lumen as a response to feeding.

Based on suggestions that the hepatopancreas tubules would be filled with digestive enzymes and chyme during digestion, it was believed that the cross-sectional area of the lumen, as a percentage of tubule area, would expand and contract in response to the movement of fluids. However, this study shows that there was no significant change in lumen area, which suggests that the fluid flow (enzyme secretions or chyme) within the lumen of the tubules was either constant, or the rate of flow was not strong enough to cause a significant change in lumen area. A constant fluid flow within the tubule lumen is indicative that enzyme production was likely to be continual.

Digestive Enzymes of Fed Versus Unfed Animals Over Time

It has been reported that feeding triggers enzyme production in decapods (Barker & Gibson 1977, Barker & Gibson 1978, Al-Mohanna et al. 1985a, Vogt et al. 1989), as shown by higher enzyme activities in the fed animals over time. However, these authors did not conclusively demonstrate the influence of feeding on enzyme production, because starved controls were not used, so it is possible the enzymatic response may be because of disturbance at feeding rather than the feeding event itself. In this study, we did not find any evidence from the enzyme analyses to conclude that P. monodon also displayed a positive digestive response to feeding.

We found that there was no significant differences in amylase activity between fed and unfed animals, or alternatively that trypsin and [alpha]-glucosidase activities in the fed animals were significantly lower than those in the unfed animals. The increased trypsin and [alpha]-glucosidase activity in unfed animals could be because of starvation. Prawns have been shown to use carbohydrate, lipid and protein reserves sequentially during starvation (Cuzon et al. 1980), which may be depleted after approximately 2 days of starvation (Stuck et al. 1996). Therefore, it is possible that the extended starvation period imposed on the unfed animals during the experiment's preparation in this study could thus have caused the increase in trypsin and [alpha]-glucosidase activity.

Hernandez-Cortes et al. (1999) also found no significant difference in trypsin activity between fed and unfed P. vannamei. This lack of significant difference in trypsin activity as demonstrated by Hernandez-Cortes et al. (1999), supports our findings that feeding did not have an influence on enzyme production, which would be indicated by an increase of trypsin in fed animals.

In this first examination of lipase activities in P. monodon, it was found that lipase activity peaked at 60 min post feeding, which was twice the activity at 480 min post feeding (Fig. 3). There was also a decreasing trend of enzyme activities over time observed in the [alpha]-glucosidase assay (Fig. 2). Both these trends are common for fed and unfed animals, because of the lack of significance in the orthogonal ANOVA, and they suggest that this trend may be caused by disturbance and not actual provision of feed.

Comparison of Examined P. monodon Enzyme Activities With Other Decapods

The trypsin activities in P. monodon were similar to reported activities in most penaeids (Table 4). The higher trypsin activity in C. maenus is probably caused by its mainly carnivorous diet. Trypsin activity is influenced by the amount of protein and protein source in the diet. This was demonstrated by Lee et al. (1984), who found that P. vannamei fed a diet with a 38% protein inclusion gave a significantly higher protease activity. Smith et al. (1985), found similar results with P. vannamei when the diet used had a 36% protein inclusion. Because the amount of protein in the commercial prawn pellets used in this study (39%) was similar to the protein inclusions in the diets used by Smith et al. (1985) and Lee et al. (1984), it could be assumed that the trypsin activity generated by P. monodon in this study was efficiently assimilating the protein in the diet.

Smith et al. (1985) also found that the protein source was more substantial than protein amount on influencing growth in medium and large animals (mean weights 9.8 g and 20.8 g respectively). Rodriguez et al. (1994) made a similar suggestion that trypsin activity could be influenced by diet. In their study, P. japonicus feeding on the algae Chaetoceros gracilis had six times more trypsin than animals feeding on Artemia nauplii (Rodriguez et al. 1994). Thus, the type of proteins in the diet must also influence the trypsin activity. Because trypsin activity has been found in P. monodon in this study, it could also be safely assumed that other proteases like carboxypeptidases were present in P. monodon to complete protein digestion. These other enzymes, although not examined, were found to be present in other penaeids (Lee et al. 1984).

The [alpha]-amylase activity in P. monodon differed from the reported [alpha]-amylase activities in other penaeids (Table 4) and could be explained by different experimental treatments as well as genetic differences between species.

[alpha]-Glucosidase is necessary for the final liberation of glucose residues from oligosaccharides that have been formed from the hydrolysis of large carbohydrates by [alpha]-amylase. The low activity of [alpha]-glucosidase from P. monodon studied here was lower than reported activities (Table 5), but was likely to be sufficient for complete carbohydrate hydrolysis. It seems that penaeids have naturally low [alpha]-glucosidase activities, because nearly negligible [alpha]-glucosidase activities were also found in P. indicus and P. vannamei (Omondi & Stark 1995, Le Chevalier & Van Wormhoudt 1998).

Past research has also shown a crustaceans' ability to digest carbohydrates varies with the type of carbohydrates provided in the diet (Van Wormhoudt & Favrel 1988). Generally better growth has been attained in crustaceans with the use of complex carbohydrates than with simple mono or disaccharides (Van Wormhoudt & Favrel 1988). Because there is a wide range of carbohydrates found in naturally occurring food (Kristensen 1972), it seems appropriate that prawns would exhibit a diverse carbohydrase profile to exploit the range of dietary carbohydrates (Wigglesworth & Griffith 1994). The carbohydrases examined in this study are just two of the many carbohydrases described by other authors (Wigglesworth & Griffith 1994).

Deering et al. (1996) examined lipase activity in P. monodon but were not able to quantify the lipase activity, as it was only demonstrated with the use of a triolein/agar emulsion screening (Table 5). Lipase activity was also found in other penaeids as reviewed by Jones et al. (1997) but was expressed as percent occurrence. There is currently a limited amount of literature on lipase activity in penaeids. As indicated by Le Vay et al. (2001), there should be more studies on lipid hydrolysis because current focus has been on penaeid proteases.

CONCLUSION

This study is the first to investigate the effect of feeding on the digestive enzyme production of decapods by using histological and quantitative enzymatic techniques. There were no trends that suggest feeding had an effect on the structure of the hepatopancreas or enzymatic activity. The morphological changes in the F-cells of other crustaceans examined by Barker and Gibson (1977, 1978), Al-Mohanna et al. (1985a) and Vogt et al. (1989) were not observed in this study. The lack of structural changes in the F-cells was further supported by the lack of conclusive results from the enzyme analyses. The commercial prawn feed used in this experiment contained all the necessary nutrients required by the prawns. Therefore it is expected that all the enzymes tested would be produced for proper digestion of the feed. Perhaps subsequent studies using monoingredient diets would be able to ascertain individual enzyme dynamics. In summary, the results from this study suggest a continuous enzyme production in P. monodon, which occurred even in the absence of food. These findings could be further confirmed by additional research with the use of continuously fed control animals.

ACKNOWLEDGMENTS

The authors thank Rocky Point Prawn Farm, Woongoolba, Queensland, Australia for supplying the prawns for this study and Dr. Natalie Moltschaniwskyj for her assistance in the statistical analyses.

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BOON LIONG ONG (1) AND DANIELLE JOHNSTON (2) *

(1) School of Aquaculture, University of Tasmania, Locked Bag 1-370, Launceston, Tasmania, 7250, Australia; (2) WA Fisheries and Marine Research Laboratories, PO Box 20, North Beach, Western Australia 6020, Australia

* Corresponding author. E-mail: djohnston@fish.wa.gov.au
TABLE 1.

Experimental times for feeding/disturbance and sacrifice.

 Time When Fed Time When Unfed
 Time Animals Were Sampled Animals Were Sampled

 0 min Immediately after Immediately after
 feeding event disturbance
 30 min 30 min after feeding 30 min after disturbance
 event
 60 min 1 h after feeding event 1 h after disturbance
120 min 2 h after feeding event 2 h after disturbance
240 min 4 h after feeding event 4 h after disturbance
480 min 8 h after feeding event 8 h after disturbance

TABLE 2.

The mean specific activities ([micro]mol [min.sup.-1] mg protein) of
the enzymes from the hepatopancreas of fed and unfed P. monodon
juveniles, with standard error and coefficient of variation.

 Trypsin [alpha]-Amylase

Fed
 mean [+ or -] SE 2.61 [+ or -] 0.64 18.41 [+ or -] 1.9
 CV 1.27 0.54
Unfed
 mean [+ or -] SE 5.06 [+ or -] 1.84 25.48 [+ or -] 26.72
 CV 1.92 5.55

 [alpha]-Glucosidase Lipase

Fed
 mean [+ or -] SE 0.6 x 10-3 [+ or -] 4.88 0.39 [+ or -] 0.03
 x [10.sp.-8]
 CV 3.9 x [10.sup.-4] 0.38
Unfed
 mean [+ or -] SE 1.4 x [10.sup.-3] [+ or -] 0.37 [+ or -] 0.01
 2.8 x [10.sup.-8]
 CV 1.04 x [10.sup.-3] 0.18

TABLE 3.

Interactions between and among factors for the 4 examined enzymes.

 Interaction
 Enzyme (Time x Treatment)

Trypsin F = 0.592, df 5, 43, P = 0.706
[alpha]-Amylase F = 0.799, df 5, 43, P = 0.557
[alpha]-Glucosidase F = 0.417, df 5, 43, P = 0.834
Lipase F = 0.462, df 5, 43, P = 0.802

 Enzyme Time

Trypsin F = 0.409, df 5, 43, P = 0.84
[alpha]-Amylase F = 0.376, df 5. 43, P = 0.863
[alpha]-Glucosidase F = 2.853, df 5, 43, P = 0.026
Lipase F = 3.21, df 5, 43, P = 0.015

 Enzyme Fed Versus Unfed

Trypsin F = 11.741, df 1, 43, P = 0.001
[alpha]-Amylase F = 3.884, df 1, 43, P = 0.055
[alpha]-Glucosidase F = 9.998, df 1, 43, P = 0.003
Lipase F = 0.08, df 1, 43, P = 0.927

TABLE 4.

Comparison of trypsin and [alpha]-amylase activities between Penaeus
monodon (fed and unfed) and other penaeids. Animals were in the
intermolt phase and were postlarval or juveniles. The units were
reported in units [mg.sup.-1] protein, where units are [micro]mol
[min.sup.-1].

 Trypsin

 Species Activity Method

P. monodon 2.61 Spectrophotometric assay,
 (fed) N-[alpha]-benzoylarginine-
 [rho]-nitroanalide
P. monodon 5.06 Spectrophotometric assay,
 (unfed) N-[alpha]-benzoylarginine-
 [rho]-nitroanalide
P. californiensis
P. indicus 0.62 Spectrophotometric assay,
 [alpha]-N-benzoyl-l-arginine-
 [rho]-nitroanalide
P. indicus
P. japonicus 12 * Spectrophotometric assay,
 N[alpha]-[rho]-toluenesulphonyl-
 L-arginine methyl ester
P. mulleri 0.9 Spectrophotometric assay,
 N-benzoyl-DL-arginine
 [rho]-nitroanalide
P. vannamei 0.072 Spectrophotometric assay,
 benzoyl-DL-arginine-[rho]-
 nitroanilide
P. vannamei
Litopenaeus 9.3 Spectrophotometric assay,
 vannamei N-[alpha]-p-toluenesulphonyl-
 U-arginine methyl ester

 [alpha]-Amylase

 Species Activity Method

P. monodon 18.41 Spectrophotometric
 (fed) assay,
 Ethylidene-[rho]NP-G7
P. monodon 25.48 Spectrophotometric
 (unfed) assay,
 Ethylidene-[rho]NP-G7
P. californiensis 16 * Spectrophotometric
 assay, 1% starch
P. indicus
P. indicus 0.5 * Spectrophotometric
 assay, 1% starch
P. japonicus 8 * Spectrophotometric
 assay, 1% w/v starch
P. mulleri
P. vannamei
P. vannamei 0.15 * Spectrophotometric
 assay, 1 % starch
Litopenaeus
 vannamei

Species References

P. monodon This study
 (fed)
P. monodon
 (unfed)
P. californiensis Vega-Villasante et al, 1993
P. indicus Honjo et al, 1990
P. indicus Omondi and Stark, 1995
P. japonicus Rodriguez et al, 1994
P. mulleri Fernandez Gimenez et al, 2001
P. vannamei Lee et al, 1984
P. vannamei Omondi and Stark, 1995
Litopenaeus Puello-Cruz et al, 2002
 vannamei

* Denotes figures that were estimated from values reported in graphs.

TABLE 5.

Comparison of [alpha]-glucosidase and lipase activities between Penaeus
monodon (fed and unfed) and other penaeids. Animals were in the
intermolt phase and were postlarval or juveniles. The units were
reported in units [mg.sup.-1] protein, where units are
[micro][mol.sup.-1]

 [alpha]-Glucosidase

 Species Activity Method

P. monodon (fed) 0.6 x [10.sup.-3] Spectrophotometric assay,
 [rho]-nitrophenol
 [alpha]-D-glucopyranoside
P. monodon 1.4 x [10.sup.-3] Spectrophotometric assay,
(unfed) [rho]-nitrophenol
 [alpha]-D-glucopyranoside
P. indicus 0.005 * Spectrophotometric assay,
 [alpha]-para-nitrophenol
 glucopyranoside
P. monodon
P. vannamei 0.07 Spectrophotometric assay,
 [rho]-nitrophenol-[alpha]-D
 glucopyranoside
P. vannamei 0.005 * Spectrophotometric assay,
 [alpha]-para-nitrophenol
 glucopyranoside

 Lipase

 Species Activity Method

P. monodon (fed) 0.39 Spectrophotometric
 assay, 4-[rho]-nitrophenol
 caproate
P. monodon 0.37 Spectrophotometric
(unfed) assay, 4-[rho]-nitrophenol
 caproate
P. indicus
P. monodon ([dagger]) Triolein/agar emusion
 clearing
P. vannamei
P. vannamei

 Species References

P. monodon (fed) This study
P. monodon
(unfed)
P. indicus Omondi and Stark, 1995
P. monodon Deering et al, 1996
P. vannamei Le Chevalier and Van
 Wormhoudt, 1998
P. vannamei Omondi and Stark, 1995

* Denotes figures that were estimated from values reported in graphs.

([dagger]) Represents a positive reaction using a triolein/agar
emulsion screening.
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Author:Johnston, Danielle
Publication:Journal of Shellfish Research
Geographic Code:1USA
Date:Apr 1, 2006
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