Digestive Physiology of Mud Crab Scylla Serrata Forskal

CERTIFICATE OF ORIGINALITY

I hereby declere that this submission is my own work and that, to the belt of my knowledge and belief, it contains no material previously published or written by another parson nor material which to a substantial extent has been accepted for tha award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in tha text.

(Signed) DIGESTIVE PHYSIOLOGY OF MUD CRAB SCYLLA SERRATA FORSl

by PURNAMA SUKARDI

A thesis submitted to the University of New South Wales for the degree of Doctor of Philosophy

School of Biological Science, University of New South Wales, November, 1993. STATEMENT

I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text. CONTENTS

LIST OF TABLES ...... p.iv

ABSTRACT ...... p.1

GENERAL INTRODUCTION ...... p.3

THE DIGESTIVE SYSTEM OF CRUSTACEA ...... p.3

DIGESTION Mechanical digestion ...... p.3 Chemical digestion ...... p.4

DIGESTIVE ENZYMES Proteases ...... p.4 Li pases ...... p.6 Carbohydrases ...... p. 7

NUTRIENT REQUIREMENTS OF CRUSTACEANS ...... p.8

BIOLOGY OF SCYLLA SERRATA FORSKAL ...... p.10

ASSIMILATION OF FOODSTUFFS IN CRUSTACEA ...... p.12

ASSIMILATION STUDY ...... p.15

INTRODUCTION ...... p.15 General Background ...... p.15 Dietary protein ...... p.15 Dietary lipid ...... p.16 Dietary energy ...... p.18 Assimilation-Background ...... p.18 Assimilation Methodology and Term ...... p.19

MATERIAL AND METHODS ...... p.21

GENERAL MATERIALS AND METHODS ...... p.21 Apparatus for assimilation and enzymatic study ...... p.21 Apparatus for the rate of passage study ...... p.21 Water analyses ...... p.22

MATERIALS AND METHODS FOR ASSIMILATION STUDY ..... p.22 Experimental animals ...... p.22 Dietary treatments ...... p.22 Sample collection ...... p.23 Sample storage ...... p.23 Analytical procedures ...... p.23 Protein and Kjeldahl ...... p.23 11

Fat ...... p.24 Gross energy ...... p.24 Ash ...... p.25 Chitin digestion ...... p.25

Measurement of assimilation ...... p.25

Statistical analyses ...... p.26

RESULTS p.27 Composition of experimental diet ...... p.27 Rate of nutrient intake and output ...... p.28 Assimilation of experimental diets ...... p.28

DISCUSSION ...... p.31 Dry matter ...... p.31 Protein ...... p.31 Assimilation of protein ...... p.32 Lipid ...... p.33 Energy ...... p.34 P:E ratio ...... p.35 Assimilation of chitin ...... p.36

RATE OF PASSAGE STUDY ...... p.39 INTRODUCTION ...... p.39

MATERIALS AND METHODS ...... p.40

PARTICLE MARKER ...... p.40 Experimental apparatus ...... p.40 Experimental animals ...... p.40 Dietary treatment for particle and soluble marker experiments . . . . . p.40 Marker used ...... p.41 Collection and storage of samples ...... p.41 Radioisotope counting ...... p.41

SOLUBLE MARKER ...... p.42 Marker used ...... p.42 Collection of samples ...... p.42 Sample analyses ...... p.43 Data analyses ...... p.44 Statistical analyses ...... p.44

RESULTS ...... p.45 Particle marker ...... p.45 Fluid marker ...... p.45

DISCUSSION ...... p.47 Particle marker ...... p.47 Fluid marker ...... p.48 iii

ENZYMATIC STUDY ...... p.50

INTRODUCTION ...... p.50

MATERIALS AND METHODS ...... p.53 Experimental apparatus ...... p.53 Dietary treatment ...... p.53 Preparation of substrate for chitinase assays ...... p.53 Buffers for assays ...... p.54 Sample collection and preparation for NAGase and chitinase assays . p.54 Collection foregut juice ...... p.54 Preparation of foregut juice ...... p.55 Preparation of digestive gland extracts ...... p.55

NA Gase ASSAY ...... p.55 Effect of pH on absorption at 410 nm by p-Nitrophenol ...... p.57

CHITINASE ...... p.57

CALCULATION OF~ AND Vmax ...... p.58

STATISTICAL ANALYSES ...... p.59

RESULTS p.60 The pH of foregut juice ...... p.60 The effect of feeding and fasting on chitinolytic activity of foregut fluid p.60 Kinetic properties of chitinolytic enzymes ...... p.60 The effect of pH on enzyme activity ...... p.62 The effect of temperature on enzyme activity ...... p.63 Activity of chitinolytic enzymes in the midgut gland ...... p.64

DISCUSSION ...... p.65 pH of foregut juice ...... p.65 Feeding and fasting crabs ...... p.65 Kinetic properties ...... p.66 Effect of temperature ...... p.67 pH of digestion ...... p.70 Enzyme activity in the mid gut gland ...... p.72

GENERAL SUMMARY ...... p.75

CONCLUSIONS ...... p.77

ACKNOWLEDGEMENTS ...... p. 78

REFERENCES ...... p.79 IV

LIST OF TABLES

1. The composition of diet fed to the crabs ...... p.27

2. Daily intake and output of nutrients and energy in experimental diets ..... p.29

3. A comparison of apparent assimilation coefficient (AAC) of dry matter from some decapod crustaceans ...... p.32

4. Assay mixtures for PEG determination ...... p.43

5. Cumulative particle marker excretion in Scylla se"ata ...... p.45

6. Comparation of cumulative marker excretion in Scylla serrata and other species ...... p.48

7. Assay mixture for determining NAGase activity ...... p.57

8. Assay mixture for determining chitinase activity ...... p.58

9. The pH and properties of the foregut juice ...... p.61

10. Kinetic properties of the chitinolytic enzymes in Scylla serrata ...... p.62

11. Spesific activity of NAGase and chitinase in the digestive gland of Scylla se"ata ...... p.63

12. Comparison of Michaelis constants from some arthropods ...... p.68 1 ABSTRACT

This study investigated the rate of passage and assimilation of foodstuffs and the role of chitinolytic enzymes in the digestion of natural diet in Scylla sen-ata.

Assimilation of nutrients and energy from the diet were measured using direct methods and expressed as apparent assimilation coefficients (AAC). On diets of bivalves (Plebidonax deltoides) or prawns (Metapenaeus macleayi), measured AAC were high for all components measured. Thus AAC for crude protein was 98.07%

(bivalve) to 98.64% (prawn); for crude lipid 94.3% (bivalve) to 95.94% (prawn) and

91.05% (bivalve) to 93.12% (prawn) for total dry matter. Similarly high values were obtained for energy derived from these sources. The results showed that protein, lipid and total content from both diets were highly assimilated and protein was the major energy source available to the crabs. Assimilation of chitin in the diet was also very high.

The rate of passage of particulate matter through the digestive tract was measured using an indigestible marker {57Co labelled microspheres).

Polyethyleneglycol (PEG) and 51 Cr-EDTA were investigated as markers for the fluid fraction of the digesta. The mean retention time of particle marker was 19h. 51 Cr

EDTA was absorbed across the gut of Scylla and is not useful as a fluid marker.

PEG was not absorbed and was thus potentially useful in this role. However, the rccirculatory pattern of fluid digesta in Scylla renders the use of fluid markers inappropriate ( only 12% of the marker was excreted after 648h).

Methods were developed for the assay of chitinase and {3-N acetylglucosaminidasc (NAGase) in the digestive tract of Scylla. The apparent K.n value was 0.11 ± 0.06 mmol.L1 for NAGase and 4.32 ± 2.65 mg· 1 chitin.mL1 for chitinasc. Activities of both NAGase and chitinase were increased by raising the temperature. up to the maximum used (30"C). Activity was strongly pH dependent 2 and peaked for both enzymes at pH 8. Normal pH in the digestive juice was 6.06 ±

0.2 (feeding crab) and 5.5 ± 0.1 (fasting crab). The enzyme activities were not altered by short periods without food. 3

GENERAL INTRODUCTION

The Digestive System of Crustacea

The Crustacea are a predominantly aquatic group, with about 42,000 described species of which crabs comprise 8,500 species (Gardiner, 1972; Meglitsch, 1972;

Barnes, 1987). The digestive system of crustaceans is composed of the alimentary tract and several associated diverticula. The crustacean gut is usually a straight tube which is divided into foregut, midgut and hindgut. The foregut (stomodaeum) is ectodermal in origin and lined with cuticle. It is subdivided into the oesophagus, cardiac stomach and pyloric stomach. In most malacostracans, the cardiac stomach has a gastric mill which consists of three calcareous teeth (two are lateral in position and one is dorsal) whilst the pyloric stomach is a filtration device. The midgut

(mesenteron) arises from endodermal tissue and is not lined with cuticle. The hindgut (proctodaeum) is ectodermal in origin and lined with cuticle (Lockwood,

1968; Van Wee!, 1970; McLaughlin, 1983).

Digestion

Mechanical digestion

Digestion by crustaceans, includes a mechanical phase involving peristalsis and trituration and a chemical component enabled by enzymes and other chemical factors in the digestive juice~ (Vonk, 1960). The chelae and mouthparts are used to reduce the food to small particles which enter the mouth and are mixed with secretions of the salivary glands. Food is then swallowed and passed via the short oesophagus to the cardiac stomach. Here the gastric mill grinds it into pieces fine enough to pass 4 through the sctal filter of the pyloric stomach (Meglitsch, 1972; Gardiner, 1972;

Warner, 1977). This mechanical breakdown is supplemented by the digestive juice, produced by the midgut gland, which contains a wide range of digestive enzymes including proteases, lipases, and carbohydrases (Jennings, 1972; Morris, 1991 ).

Chemical digestion

Enzymes are the principal chemical components in digestion. Digestion has been reported to be influenced by the hydrogen-ion concentration (pH) of the gut. Whilst most crustacean guts arc slightly acid, an alkaline digestive juice has been recorded in some species. Little is known concerning the source of the ions and molecules responsible for the pH of the gut contents (Van Weel, 1970; Barker and Gibson,

1979; Bade, 1974).

Digestive Enzymes

A number of proteolytic enzymes, amylases and lipases have been identified in extracts of midgut gland tissues and in the digestive juice of decapod crustaceans

(Vonk, 1960; Gibson and Barker, 1979; Dall and Moriarty, 1983). They have capacity

to digest a wide range of foodstuffs including complex carbohydrates such as chitin

and cellulose.

Proteases

Proteolytic enzymes (proteases, peptide hydrolases) have been studied extensively

in the Crustacea, especially the decapods (Van Weel, 1970; Amstrong and DeVillez,

1978). Proteases arc enzymes which hydrolyse peptide bonds in proteins. They are

divided in two major groups; endoproteases which attack peptide bonds within the

protein chain. while cxoproteases cleave peptide bonds at either end of a peptide

chain (Lehninger, 1975; Ladd Prosser and DeVillez, 1991; Mahler and Cordes, 1971). 5

The endoproteascs of decapod crustaceans include trypsin, chymotrypsin, capthesin and collagenase, whereas exoproteases comprise carboxypeptidases, aminopeptidases and dipeptidases (Gibson and Barker, 1979). Protease activity was greater than that of amylase and lipase in Homarus americanus and had an optimum pH in the range 5.5-7.5 (Hoyle, 1973). A cysteine protease was identified from the digestive juice of H. americanus and found to have a molecular weight of 28,000, with a peak of activity at between pH 4 and 5 (Laycock, et al. 1989).

Among the endoproteases, trypsin-like activity has been reported in a number of

malacostracans including the decapods Orconectes virilis (De Villez, 1965), Penaeus kerathurus, P. japonicus (Galgani, et al. 1984), and Panulirus japonicus (Galgani and

Nagayama, 1987), the amphipods, Synurella and several species of isopods e.g. Jdotea,

Exasphaeroma, Limnora and Lirceus. (Buchlen, 1967; DeVillez, 1968; Lu, et al. 1990).

In Penaeus setiferus, the enzyme has a molecular weight of 24000 and the optimum

pH was in the range of pH 7.0-9.5 (Gate and Travis, 1969). It seems likely that

trypsin-like enzymes are characteristic of malacostracans.

Chymotrypsin is commonly present in decapods and has been found in the

digestive gland and digestive juice of numerous species e.g. Cancer irroratus, C.

borealis (Brun and Wojtowicz, 1976) and Homarus americanus (Brockerhoff, et al.

1970). In Vea pugilator it has been observed in the midgut gland (Eisen and Jeffrey,

1969). In Penaeus monodon, P. penicillatus, P. japonicus, Metapenaeus monoceros and

Macrobrachium rosenhergii, it has an optimum pH ranged between 7 to 8 (Tsai, et al.

1986 ). On the other hand. this enzyme was not demonstrated in either Panulirus

japonu:us ( Galgani and Nagayama. 1987) or Orconectes virilis (De Villez, 1965).

Collagcnase activity has also been detected in decapods e.g. the fiddler crab, Vea

pugilator (Eisen and kffry. 1969) and Penaeus monodon (Lu, et al. 1990). Purification 6 and characterization of this enzyme indicated a pH optimum of 8, similar to that of trypsin- and chymotrypsin-like enzymes. The molecular weight was about 25000 and the composition of amino acids was similar as trypsin- and chymotrypsin, as well

(Eisen, et al. 1973). In the japanese lobster, Panulirus japonicus the molecular weight of collagenase was 24000 and the optimum pH was about 8 (Galgani and Nagayama,

1987).

Exoproteases have been found in many decapod crustaceans. Carboxypeptidases

A and B are present in the midgut gland of prawns,Penaeus kerathurus , P. japonicus

(Galgani, et al. 1984) and P. vannamei (Lee, et al. 1984). In Penaeus setiferus, they have a molecular weight of 34200 and 30000, respectively (Gate and Travis, 1973).

Carboxy peptidase A was found in Panulirus japonicus (Galgani and Nagayama, 1987) and Homarus americanus (Brockerhoff, et al. 1970), but carboxypeptidase B was not demonstrated in either species.

Aminopeptidases have been recorded from decapods. Leucine aminopeptidase activities were identified in Panulirus japonicus (Galgani and Nagayama, 1987),

Penaeus kerathurus and P.japonicus (Galgani, et al. 1984). However, they were not detected in the crayfish, Orconectes virilis (DeVillez, 1965) and Homarus americanus

Brockerhoff, et al. 1970).

Zymogens are inactive precursor of proteolytic enzymes (Lehninger, 1975).

They were not identified in Homarus americanus (Brockerhoff, 1970) and Penaeus japonu:us ( Gate and Travis, 1969) nor in a range of other crustaceans (Dall and

Moriarty, 1983). Histochemical studies, however, identified zymogens in the prawn,

Penaeus semisulcatus (AI-Mohana, et al. 1985) where they were produced and secreted by F-cells of the midgut gland. Zymogen granules are released in the first two hours after a meal.

Lipases 7

Lipases activities have been carried out in many decapod crustaceans with crude extracts or homogenates of midgut gland tissue or gut content and digestive juice. In some cases whole animals have been homogenized (Van Wee!, 1970). A lipases was identified in the digestive juice of Homa,us americanus, with a molecular weight of

43000 and an optimum pH about 7 (Brockerhoff, et al. 1970). In young H. americanus, the highest activity of this enzyme was found at 36 d of age (Biesiot and

Capuzzo, 1990). In H. gamma,us and Panuli,us elephas, lipases appear to split fats better than they do esters (Vonk, 1960). Tributyrinase activities were observed in the digestive gland of the Jonah crab, Cancer borealis and rock crab, C.irroratus.

Tributyrinase activities from C. irroratus was higher than that of C. borealis (Brun and

Wojtowicz, 1976).

Carbohydrases

Amylases are of widespread occurrence in crustaceans and a-amylase is the major amylase in this group (Vonk, 1960). It has been isolated from Uca minax, U pugnax

and U. pugilator and found to have a pH optimum at 7.3 (Azzalina and Trainer,

1985). The activity of amylolytic enzymes have been found to increase during development of larval stages in H. americanus (Biesiot and Capuzzo, 1990). Amylases

activity of the digestive juice of fasted H. americanus was significantly lower than in

feeding crabs and a hroad activity was found from pH 4 to pH 7, with a peak at 5.5

(Hoyle. 1973).

Cellulase activities have been demonstrated in the digestive juice and midgut

gland of crustaceans (Yokoe and Yamasu, 1964; Elyakova, 1972). The activity of

cdlulase in gut extracts of crustaceans was higher than that from insects (Monk,

I 976 ). The cdlulosc digestion took place in the proventriculus, midgut gland and

anterior midgut of the freshwater amphipod, Gamma,us pulex and found to have an 8 optimum pH at about 5.0-6.5 (Monk, 1977).

Chitinases have been observed in the digestive tracts of crustaceans (Brockerhoff, et al. 1970; Elyakova, 1972). Chitin digestion has also been examined in the juvenile

Penaeus vannamei, adult P.setiferus, juvenile P. duorarnm (Clark, et al. 1993) and adult red swamp crayfish, Procambarus clarldi (Brown, et al. 1986).

Nutrient Requirements of Crustaceans

The natural diets and the nutritional requirements of the Crustacea have been studied extensively, especially for the decapod crustaceans (Ruello, 1973; Conklin,

1980; Paul, 1981; Wear and Haddon, 1987; Dall, et al. 1991 ). Crustacea require amino acids, lipids, carbohydrates, vitamins and minerals in their diet for normal growth, maturation and general metabolism (Dall and Moriarty, 1983).

Several studies have been conducted to establish the requirements for amino acids of decapod crustaceans to enable formulation of diets suitable for intensive culture (Thompson and Farragut, 1966; Hird, 1986; Akiyama, 1989). Many species require specific amino acids in the diet e.g. the prawn, Palaemon serratus is unable to synthesize some amino acids and must obtain them from their diet (Cowey and

Forster, 1971 ). In Paneus aztecus nineteen amino acids are present in the tissues and eleven of which are considered to be essential (Shewbart, et al. 1972). A diet containing methionine-enriched soybean plastein for Penaeus japonicus was found to promote growth, and increased efficiency of utilization of food and protein, but supplementation with crystalline methionine or soybean-plastein only in the diet had no effect on growth and utilization efficiencies (Teshima, et al. 1992).

Linoleic (n6 family) and linolcnic (n3 family) fatty acids are essential for most animals ( Morris, 1991) and either linoleic or linolenic fatty acids have been shown to be essential to many crustaceans. including crabs, lobsters and prawns (McConaugha, 9

1979). The prawn, Penaeus japonicus can convert linolenic acids (18:3~3) to poly unsaturated fatty acids (linoleic acids: 20:5w3 and 20:6w3). However, the alteration was too slow to meet the requirements solely by this route. Thus the prawn requires some dietary polyunsaturated fatty acids to achieve maximum growth and survival

(Jones, el al. 1979; Kanazawa, el al. 1979).

The larvae of the mud crab, Rhithropanopeus harrisii and the spider crab Libinia emarginata can not synthesize sterols acetate and these must be obtained from their diet (Whitney, 1969; Dadd, 1970). Cholesterol is the principal constituent of neutral lipids in some prawns and is used for normal growth and metabolic maintenance

(Teshima, el al. 1977; Guary and Kanazawa, 1973).

Carbohydrates comprise about 2% of the body weight of juvenile lobster,

Homarus americanus (Capuzzo and Lancaster, 1979). The requirement for carbohydrates has been observed to be parallel with the growth of juvenile crayfish,

Astacus astacus. Increasing body size of the juvenile was followed by increasing dietary carbohydrate digestion requirement (Ackeffors, 1992). Carbohydrates can be used to substitute for dietary protein in Penaeus monodon without a reduction in weight gain, survival rate or feeding efficiency (Shiau and Peng, 1992). The type of carbohydrate fed to the prawn, Penaeus monodon influenced the weight gain (Aiava and Pascual, 1987).

Some vitamins, including vitamin B (thiamin), Vitamin B2 (riboflavin), nicotinamide (niacin). pyridoxine, pantothenic acid, folic acid and biotin, appear to he essential for crustaceans (Dall and Moriarty, 1983). The level of dietary vitamin B required for good growth in Penaeus monodon was 14 mg.kg·1 diet based on haemolymph blood thiamin analyses. Lack of this vitamin resulted in retarded growth. poor food conversion ratio and low survival rates (Chen, et al. 1991 ). When

P. vannamei was fed a diet without supplementation of vitamin E subsequent growth 10 was retarded and greater mortality was observed (He, et al. 1992).

The requirement~ for some minerals have been studied in decapod crustaceans

(Gibson and Barker, 1979; Conklin, 1980; Dall and Moriarty, 1983). The midgut gland appears to be the site of absorption, storage, metabolism and detoxification of minerals. Calcium and magnesium are required in postmoult by the blue crab,

Callinectes sapidus (Vigh and Dendiger, 1982) and this is probably true of most crustaceans. Dietary intake of copper is required by juvenile Penaeus vannamei for the best growth and tissue mineralizations and a diet containing 0.5 mol Cu.kg-1 food is considered to be optimal (Davies, et al. 1993). The requirement of phosphorus in the diet has been observed in P. vannamei (Civera and Guillaume, 1989). A diet with a Ca:P ratio of 0.5 gave optimal growth and survival in Homarus americanus

(Gallagher, 1978).

Biology of Scylla se"ata Forskal

Scylla sen-ala belongs to the Portunidae (swimming crabs)in which the last pair of legs is modified as swimming paddles and nine even-sized teeth are present on each side of the body behind the eyes (Warner, 1977; Fielder and Heasman, 1978). Scylla serrata exists in several colour varieties which have been given different names. Scylla sen-ala is deep ferruginous brown to light purplish brown in colour, whilst in S. sen-ala var. paramamosain the colour is lighter. S. oceanica is green to greyish green and S. lranquebarica ( deep purplish drab-green) (Estampador, 1949). However, all these various colour forms have been synonymised and are now regarded as a single species (Stephenson and Campbell, 1959). The distribution of Scylla extends from

East Africa and the Red Sea to Japan, Tahiti, New Zealand and Australia. Within

Australia distribution l'Xtends from Western Australia to Darwin, Queensland and

Nl:'w South Wales (Stephenson and Campbell, 1959). 11

Scylla is a favoured dietary item and is subjected to heavy fishing pressure throughout its Inda-Pacific range. Currently its mariculture is receiving considerable attention. In South East Asia, overfishing has threatened the viability of this fishery, and attention has been given to the possibility of its culture (Bardach, 1972). In

Australia, the experimental culture of this crab from the hatching of eggs up to megalopa stage under laboratory conditions has been achieved but commercial rearing is not yet done (Heasman and Fielder, 1984; Queensland Department of

Primary Industries, 1984 ). In Taiwan, larvae were successfully reared throughout the zoeal stages (90%-100% survival), but cannibalism occurred during megalopal and

crab1 stages so that only 20% ultimately survived (Cowan, 1984). In the Philippines, preliminary studies on the propagation and cultivation of mud crabs in brackish water ponds have been conducted (Escritor, 1972). In Thailand, the young crab (carapace length 7-14 cm) has been reared successfully in ponds and reached marketable size

(carapace length more than 10 cm) within a period of 45 days (Varikul, et al. 1972).

There is thus considerable interest in culture of this species and information regarding digestion and assimilation of natural diets is needed as a basis for formulation of artificial diets to feed cultured crabs.

The gut of Scylla is essentially similar to that described above for crustaceans generally. The oesophagus is a short duct with many tegumental glands which produced mucous to lubricate food entry. The chitinous lining of the cardiac stomach has calcified ossicles forming the gastric mill. Here trituration occurs and food is intimately mixed with digestive juices secreted by midgut gland. The midgut is very short and lacks a chitinous lining. The hindgut extends through the abdomen and the anus is on the telson (Hill, 1976; Barker and Gibson, 1978). Histochemical studies of the epithelium of the midgut gland of tubules of decapods identify four cell types: E-( embryonic), F-(fibrillar), B-(blister-like) and R-(resorptive) cells 12

(Gibson and Barker. 1979). The midgut gland not only produces the digestive juice. which is transported forwards to the stomach and initiates digestion in this region, but also absorbs, digests and stores nutrients and excretes waste materials (Van

Wee), 1970; AI-Mohana, et al. 1985). In Scylla and Homarus americanus, midgut gland F-cells and B-cell are the source of the digestive enzymes. In Scylla the digestive enzymes arc secreted in three waves of activity 0.5-1 h, 3 h and 8 h after a meal, whilst in Homarus secretion occurred at 0-15 min, 1-2 hand 3.5-5 h. The digestion in these species is principally extracellular and the digestive cycle is reported to be complete within 12 h (Barker and Gibson, 1977; 1978).

Measurement of assimilation of foodstuffs in Crustacea

The effectiveness of digestive process is generally assessed by measuring assimilation of a particular diet. Assimilation may be defined as;

Assimilation = { ( c-:)}

Where C is food consumed and Fis faeces produced. (Grahame, 1983). Assimilation can be measured in varieties of ways including a direct method, which involves accurate recording of total food eaten and total faeces excreted by animals (Cullison and Lowrey, 1987), and indirect methods which use inert markers to achieve the same end (Maynard and Loosli, 1956). The disadvantage of the direct method is that it may required a long measurement period to collect enough faeces and the measurement of food intake and faecal output have to be very accurate. To overcome this problem, an indirect method was developed using the inorganic ash left after combustion of food and faeces as an indicator of absorption with an 13 assumption that the ash was not assimilated during digestive processes (Conover,

1966). Both methods have potential problems and these are discussed in more detail later. Factors known to influence assimilation efficiency include the type of food, body temperature, the availability of food and the length of time of food is available, morphology of the digestive tract and morphology of the food components (Conover,

1966; Dall and Moriarty, 1983). Assimilation values may vary widely between different species (Schindler, 1971).

Crabs exhibit a wide range of feeding habits and diets, but most of them are predaceous and or scavengers (Warner, 1977). Portunid crabs are mostly carnivorous and invertebrates arc common prey. For example, crustaceans and molluscs are the main food of Callinectes arcuatus and C. toxotes, from the Pacific Coast of Mexico

(Paul, 1981) and for Ovalipes catharns, New Zealand (Wear and Haddon, 1987).

Brown algae and molluscs were second in preference after crustaceans for

Liocarcinus puber, whilst crustaceans, molluscs and fish formed the main part of the diet of L. holsatus (Choy, 1986). For Scylla, molluscs were the main food item in

South African and Australian populations, whilst crustaceans comprised 22.5% and

20% of the diet, respectively (Hill, 1976).

Little is known of the dietary requirements of crabs under culture conditions. The effect of various levels of dietary protein on survival, moulting frequency and growth of juvenile blue crabs, C. sapidus, have been examined (Millikin, et al. 1980). In this species, crabs fed a diet containing either 37% or 49% crude protein were larger than those fed 23% crude protein. Animal lipids (such cod liver oil) and animals protein (like casein) were more digestible for Carcinus maenas than those of vegetable lipids (maize oils) and vegetable protein (Ponat and Adelung, 1980).

In view of the commercial importance of Scylla the current study is designed to 14 explore its ability to digest the principal components of its natural food. Secondly, the rate of passage will be measured to establish the retention time and thus required frequency of feeding under culture condition. Finally, the ability of Scylla to digest chitin is examined. Chitin intake on a natural diet is appreciable as it is a major component of crustacean exoskeleton. Chitin intake on a natural diet is appreciable as it is a major component of crustaceans exoskeleton and represent ::::: 6 % of dry matter in prawn. Strong ability to digest this material would allow inclusion of commercial crustacean waste from prawn peeling to be used in formulation of artificial diets for culture crabs. 15 ASSIMILATION STUDY

2.1. INTRODUCTION

In intensive aquaculture feeding is a major expense and the success of the venture is sensitive to the cost and nutritional quality of the diet. Substitution of expensive components of the diet of the diet ( e.g. protein) with cheaper materials, while maintaining high levels of growth, is, therefore, of considerable interest and knowledge of optimal composition of diets for cultured animals becomes important

(Lee and Wickins, 1992; OECD, 1989). One method of determining the suitability of a diet is to measure its assimilation efficiency, and this approach has been commonly used in studies of nutritional assessment of aquatic animals in recent times (Richly and Spanhoff, 1979; Lee and Lawrence, 1985).

2. 1.1. General Background

Dietary Protein

In general, crustaceans have a high requirement for dietary protein (Castell and

Budson, 1974). Proteins are a major component of the body in many crustaceans e.g. they make up about 50 % of the dry weight in the euphausiids, Euphausia superba,

Meganyctiphanes norvegicus and Nemomycis integer (Srinivasagam, et al. 1971 ). Protein composed more than 70 % muscle dry mass in Cancer magister (Allen, 1971).

To achieve good growth rates in prawn, the optimal levels of crude protein vary he tween 20-70 % (Wickins, 1976 ). Protein is required in large amount during development of Pagurus bemhardus, Carcinus maenas (Dawirs, 1980; Dawirs, 1981) and the kuruma prawn, Penaeus japonicus (Lim and Hirayama, 1993). An artificial diet containing 30<7< protein level was optimum for good growth of juveniles of

Macrobrachium rosenhergii (Koshio. 1992). 16

In addition to an adequate intake of protein, crustaceans require number of essential amino acids. In the prawn, Palaemon se"atus is unable to synthesize some amino acids and must obtain them from their diet (Cowey and Forster, 1971). In

Penaeus japonicus, supplementation of the diet with casein enhanced the growth

(Teshima, et al. 1986 ). The amino acid requirements are not the same for all crustaceans, e.g. in the tissues of Penaeus aztecus, nineteen amino acids are present, eleven of which are essential, leucine being the most important of these (Shewbart, et al. 1972).

Leucine is also most important in the blue crab, Callinectes sapidus (Thompson and Farragut, 1966) and in Cancer magister (Allen, 1971) leucine was the main essential amino acid required in the diet. Arginine is essential in the crayfish, Cherax destructor and lack of this amino acid demonstrated cannibalism (Hird, et al. 1986).

Supplementing artificial diets with arginine produced improvement in growth in the prawn, Penaeus monodon (Chen, et al. 1992). Arginine was also found to be highly assimilated by P. vannamei (Akiyama, et al. 1989).

Dietary Lipid

Lipids include triglycerides (fats), phospholipids, sterols, sterol esters, waxes, and derived lipids such as fatty acids and carotenoids (West, et al. 1966; Gunstone, 1967).

Lipids are not only a good source of energy, but are commonly the major form in which organic energy reserves are stored. They are also important in maintaining the structural and physiological integrity of cellular membranes (O'Connor and Gilbert,

1968; D'Abramo and Conklin, 1985). The fatty acids commonly found in crustaceans are palmitic acid (Cl6:O), palmitoleic acid (Cl6:lep7), oleic acid (C18:lep9), linoleic acid (C18:2ep6), linolrnic acid (C18:3ep3), arachidonic acid (C20:4ep6), dcosapt:ntanoic acid (C20:5ep3) and docosahexanoic acid (C22:6ep3) (Dall, et al. 17

1991; Kanazawa, et al. 1979; Navarro, et al. 1992). In prawns, the major saturated fatty acid is palmitic acid (16:0), whilst the linoleic ( ~-3) group comprised the main polyunsaturated fatty acids (Wickins, 1976). The midgut gland of crustaceans has major functions in the digestion and absorption and storage of lipids (Chang and

O'Connor, 1983; Gibson and Barker, 1979). Most arthropods are incapable of synthesizing unsaturated fatty acids (Chang and O'Connor, 1983) and they are essential in the diet of Homarus americanus (Castell and Cowey, 1976), Penaeus merguensis (Clark and Wickins, 1980), P. monodon (Millamena, et al. 1984), P. setiferus and P.duorarum (Bottino, et al. 1984) and P. japonicus (Kanazawa, et al.

1985; Aiava, et al. 1993 ).

Many crustaceans are incapable of synthesizing sterols e.g. the decapods,

Callinectes sapidus, Astacus astacus and Homarus vulgaris (Zandee, 1964), Panulirus japonicus and Porlunus trituberculatus (Teshima and Kanazawa, 1971) and the barnacle, Ba/anus nubilus can not synthesize squalene and sterols (Whitney, 1970).

The crab, Cancer pagurus is unable to synthesize cholesterol (Van Den Oord, 1964).

In dietary studies. cholesterol was found to be more effectively assimilated than other sterols in juvenile Penaeus japonicus (Teshima, et al. 1983). In Homarus americanus 5 % of cholesterol (as dry weight) in the diet is considered to be optimal

(Castle, et al. 1975; Zandee, 1967) whilst in Penaeus penicillatus the best growth was gained on diets supplemented with cholesterol and phosphatidylcholine at levels of ~

0.5 % and ~ 2.5 % in the diet, respectively (Chen and Jenn, 1991). The lobster is known to have the ability to synthesize phospholipids, but the capability is limited

(Shieh, 1969; D'Abramo, et al. 1981). Cholesterols are found to be effectively transferred as phosphatidylcholinc molecules ( an important component of a lipoprotein complex) from the midgut gland to the haemolymph in Homarus sp.

(D'Ahramo. ct al. 1982). 18

Dietary Energy

Energy is required to maintain normal metabolism and growth in aquatic and terrestrial animals. The major sources of dietary energy in aquatic animals and mammals are quite different. Unlike terrestrial animals, aquatic species utilize only relatively small amounts of carbohydrate and they have a low capability for digestion and metabolism of dietary carbohydrate (Love, 1988; Brett and Groves, 1979). In the prawn, Penaeus vannamei, for example, diets high in protein ( casein, gelatin, soy protein) and wheat gluten had higher assimilation coefficients than those for diets rich in carbohydrate ( corn starch). In fish, the assimilation of carbohydrate decreased when it comprised more than 25 % of the diet (Brett and Groves, 1979; Akiyama, et al. 1989). The energy that can be extracted from a diet by an animal is affected by the composition of fat, protein and carbohydrate. Different fatty acids in the diet, for example, allow different assimilation of the energy content (McLean and

Tobin, 1988). In Homarus americanus, fed with isonitrogenous diets, it was shown that the availability of energy was affected by lipids and carbohydrate (Koshio, et al.

1992).

2.1.2. Assimilation-Background

There is abundant evidence that protein is very efficiently assimilated in crustaceans. For example, in prawns, e.g. Palaemon se"atus and Panda/us platiceros, which were given commercial foodstuffs of animal and vegetable origin the digestion was very efficient (Forster and Gabbott, 1971 ). An artificial diet with 22 % protein was efficiently digested by Penaeus setiferus (Lee and Lawrence, 1985). Artificial diets containing dietary protein levels ranging from 21-61 % were highly assimilated (93-

96 o/c:) by Penaeus japonicus (Kashio, et al. 1993). An artificial diet containing 40 o/c: protein was assimilated efficiently by juveniles of the prawns, Penaeus setiferus and P. 19 aztecus (Condrey, el al. 1972).

2.1.3. Assimilation - Methodology and terms

Assimilation efficiency is an important measure of the capability of animals to digest and absorb nutrients from their diet. Measurement of assimilation efficiency has been extensively applied in studies of the nutrition of domestic and aquatic animals (Nose, 1964; Schneider and Flatt, 1974 ).

There are two principal methods for the determination of in vivo assimilation efficiencies, namely the direct methods (total collection or gravimetric method) and the indirect methods. The direct method involves measurement of total intake of nutrients in the diet and their output in faeces. The indirect method has been developed to overcome problems involved in quantitative collection by using indigestible indicators (Windell, 1978).

Many compounds have been used as indigestible markers in assimilation studies e.g. radioisotopes (Calow and Fletcher, 1972), chromic oxide (Maynard and Loosli,

1958) and inorganic ash (Conover, 1966). However the most commonly used markers in aquatic animals have been ash and chromic oxide (Furukawa and Tsukahara,

1966).

The use of inorganic material ( ash) as an indigestible marker to assess absorbtion efficiencies, assumes that only organic matter in food is affected by the digestive processes (Conover, 1966) i.e. that inorganic material is not absorbed from the food during its passage through the gut. This method has been used for many animals including sea urchins ( Hawkin, 1979), terrestrial snails (Mason,1979), the marine isopod, G/yplonolus anlarclicus (Clark, 1979) and the prawns, Penaeus setiferus and P. azlecus (Condrey. el al. 1972). However, the assumption that ash is not assimilated may not always be true and it seems to be absorbed through the gut wall in fish 20

(Tacon and Rodrigues, 1984) and in the lobster Homarus americanus (Leavitt, 1985).

Probably such absorption with subsequent excretion across gills or urine is common in aquatic animals and will result in error of measurement of assimilation.

In the chromic oxide method, the assimilation of the nutrient constituent of a diet is calculated by comparing the concentration of the indicator (which is added to the diets) in the food and in the faeces. It has been widely used in aquatic animals notably in fish e.g. Sa/mo gairdneri Richardson (Austreng, 1978), lctalurus punctatus

(Wilson and Poe, 1985), Oreochromis niloticus (Hanley, 1987) and crustaceans e.g.

Panda/us platyceros, Palaemon serratus (Forster and Gabbott, 1971) and Penaeus vannamei (Smith, et al.1985).

In fish the inert marker, chromic oxide and the direct or gravimetric methods have been compared and have demonstrated that chromic oxide diets are suitable for use in fish and provide a precise method for determining assimilation coefficients

(Wilson et al. 1981; De la Noue and Choubert, 1986). Similar tests with the crustaceans, Procambarus clarkii and Homarus americanus, however, showed that chromic oxide was absorbed through the gut wall. Thus chromic oxide is not a suitable marker for crustaceans. Gravimetric ( direct) assessment of assimilation was considered to be the better approach (Brown et al. 1986; Leavitt, 1985).

In view of this, the present study was designed to use the gravimetric or total collection method. Three experimental diets were used utilizing organisms found in the normal diet and which were readily available. In the first series, the crabs were fed with bivalves and in the second they were fed with prawns. In the third series prawns were again used, but antibiotics were included in the collection bag as a control for biological decay of faecal materials. 21

2.2. MATERIALS AND METHODS

2.2.1. General materials and methods

2.2.1.1. Apparatus for assimilation and enzymatic studies

Crabs were kept in individual plastic tanks (57cmx37cmx27cm)(volume 57 litres) supplied with flowing sea water in which salinity was maintained at 30-34. The system included three biological filter units servicing eighteen experimental tanks. Water was circulated from a tower tank to each experimental tank and returned from the sump by a float-controlled electric pump. The filter systems comprised a main filter, which refined water from experimental tanks, and two extra filters which refined water from a tower tank. The function of the extra filters was to speed up flow rates as well as refining water. Each filter unit consisted of two tanks. The composition of the first contained layers (from top to bottom) of gravel, fine sand mixed with dolomite, a combination of barbecue charcoal, gravel, fine sand and dolomite and finally gravel above perforated plastic. The layers in second tank were arranged in opposite fashion with fine sand on the top. The filters allowed a flow rate of 2.5 L.min•1 (Diagram 1).

The system was designed to maintain the following culture conditions; temperature

4 25° ± 1 C, dissolved oxygen 2:50 Torr, pH 7.2-8 and NH3 ~ 5.9 x 10 mmol.L-1.

Distilled water was added to the biofilter as needed to replaced evaporative losses.

2.2.1.2. Apparatus for the rate of passage study

The crabs were maintained individually in aerated sea water in plastic tanks

(57cmx37cmx27cm)(volume 57 litres) in a temperature and humidity controlled room at 25°C and 80 % R.H. on a 12L: 12D l)'cle. The water was changed every two days. 22

2.2.2.3. Water analyses

Conditions within the holding and experimental aquaria ( dissolved oxygen, ammonia (NH3 + NH4 +) and pH were monitored at 3 d intervals. Dissolved oxygen was measured in randomly selected aquaria using a polarographic 0 2 electrode and oxygen meter (Strathkelvin Instruments Model 781). Ammonia was measured using an Activon ammonia electrode and a Model EA 940 Ion Analyzer (Orion Research)

with NH4Cl as standards. Acid-Base analyzer Model PHM 71 (Radiometer

Copenhagen) was used to determine pH.

2.2.2. Materials and methods for the assimilation study

2.2.2.1. Experimental animals

Adult mud crabs used for the experiments, were generally bought from the Fish

Markets in Sydney, but some were caught from Middle Harbour, Sydney. Data were obtained from a total of twenty five crabs ( 456-1137 g and carapace length 7.9-9.3 cm).

2.2.2.2. Dietary treatments

Two experimental diets were used, one consisting of prawns (Metapenaeus macleayi) and the other of the bivalve (Plebidonax deltoid.es). The prawn diets consisted of prawn diet 1 and prawn diet 2. To ensure uniformity a large batch of bivalves and prawns were bought at the Sydney Fish Markets and stored frozen until used. The crabs were acclimated to the experimental conditions and the experimental diets for 7 d before measurement began. They were fed ad libitum. The food, given and the food which remained uneaten were weighed daily. The food, although stored frozen, was thawed before being given to the crabs. It was supplied daily at 1800 h. 23

2.2.2.3. Sample collection

Faecal samples were collected in a plastic bag attached over the abdomen with super glue. To remove the faeces the crabs were tied down to an expanded polystyrene board, ventral side uppermost, and the faeces were collected using forceps for solid materials and a plastic syringe for watery materials. Faeces were collected from each crab daily until the pooled sample for an individual was large enough for all the analyses (about 45 days). Antibiotics were not added to the faecal collection plastic bag in the first experiment (prawn 1 diet). In the second experiment, antibiotics were applied to check bacterial decompositions of faeces during stay in the plastic bag.

2.2.2.4. Sample storage

Food and faeces were oven-dried at 50-60°C until stable weight was attained. The dry material was finely ground using pestle and mortar and stored in a desiccator until analyzed.

2.2.2.5. Analytical Procedures

Protein and Kjeldahl N

Total nitrogen was determined in feed and faeces using a micro-Kjeldahl method

(Gerhardt Kjeldatherm Digestion System KT-4/8/12/20/40 and Vapodest 3 distillation system). Samples (.1 g) were weighed and wrapped in nitrogen free paper (Kjel-Foss

Automatic weight paper). Then sample with a Kjeldahl-tablet (1.0 g Sodium sulphate anhydrous and the equivalent of 0.05 g selenium) (Ajax chemicals) as catalyst and 5

ml H!SO4 were placed in glass tubes and digested at 420°C for 20 min in the

Gehardt Kjcldatherm digestion system. Digested samples were then distilled individually in the Vapo

, .. ,J\ . ~ mL acid( sample) - mL acid( blank) } X N ~ t ~d 1,, ,J\ N v--,mo,1 ------~~- ~---~--~ con.en s.,. v--,mo,1 mL acid( std) - mL acid( blank)

Crude protein was estimated from total N values according to the relationship

Protein= N x 6.25.

Fat

Crude fat was determined using an indirect method (petroleum ether extract) by a modification of the method given in Methods of Analyses of the Association of

Official Agricultural Chemist (-AO.AC. 1984). Samples of food or faeces (.5-2 g) were placed in extraction thimbles of known dry weight and dried in an oven at 60°C overnight. The thimble was allowed to cool in a desiccator and weighed. Fat was extracted in a Soxhlet apparatus with petroleum ether using 6-7 h extraction periods.

The thimble was again dried in an oven (l10°C) for 1 h, cooled in a desiccator and weighed to determine the weight of material (fat) extracted with ether.

Gross Energy

The gross energy content of food and faeces was determined by combustion of weighed samples (.5-1 g) in a bomh calorimeter (Gallenkamp ballistic bomb calorimeter CB 370). The temperature increase resulting from combustion within the calorimeter was measured with a thermocouple and registered on mV recorder 25

(KIPP AND ZONEN DELF BY Model BD 40). The calorimeter was calibrated by combustion of .5-1 g samples of benzoic acid (26454.3 ± 2.5 J.g-1). Feed, faeces, and standard were compressed into tablets before being ignited. Most faecal and food samples left large apparently inorganic residues. To check that full combustion had occurred these residues were finely ground and re-combustion attempted. All recombustions gave zero values. The results were corrected for the ash weight and expressed as free ash energy.

Ash

Weighed samples (.5-1 g) of food and faeces were placed in platinum crucibles in a muffle furnace at 550°C overnight. The crucible was then transferred directly to desiccator, cooled and weighed immediately to determine ash content.

Chitin

Food and faeces samples were powdered. Samples ( .5 g) were accurately weighed and digested in 20 mL of 10% KOH @ 100°C for .5 h to dissolve 'protein N'. The residual undigested material ( chitin) was washed several times with deionized water.

The chitin was then digested in 5 mL cone. sulphuric acid using the Kjeldahl digester and N content determined as described above for protein. These analyses were conducted by Bonnie Chan on samples collected by the author.

2.2.2.6. Measurement of assimilation

Many different formulae have been used to calculate the assimilation efficiency using the direct method. In principle, however, these gave similar results. For example, the nutrient retention coefficient (NRC), a formula suggested by Cowey and Seargent ( 1979) gave the same result as apparent digestibility coefficient (ADC) given by Brown. t!t al. ( 1986) and the apparent assimilation efficiem,)' (AAC) formula 26 used in this study.

NRC = Jnutrie~t r~tained} X 100 1 nutnent mtake

In this study, apparent assimilation coefficients (AAC) were estimated using the formula for the direct method given by De La Noue and Choubert (1986).

AAC= 100 { 1 _ (faecal nutrient cone. x faeces dJy weight)} ( dietary nutrient cone. x food dry weight)

Assimilation coefficients were determined for dry matter, N and protein and fat.

2.2.2. 7. Statistical analyses

Analyses of variance (ANOVA) and Honestly Significant Differences (Tukey) test were used to determine if the significance of differences in apparent assimilation coefficient (AAC) of protein, fat and energy. The Systat Software Package (Systat,

Inc. 1990) was used for testing. The criterion for significance was set at P<0.05 unless otherwise noted. All percentage data were transformed to arcsin values prior to analysis using the QUATTRO PRO Software Package (Borland, Inc.1990).

Analyses were conducted in triplicate. 27 RESULTS

Composition of Experimental Diets

Dried and powdered samples of the experimental diets were analyzed for total N

( enabling calculation of the crude protein), crude fat, chitin, gross ash, and total dry matter. Crude protein was the major constituent in all diets comprising 63.04% dry weight of the bivalves and 67.7% of the prawn diets (Table 1).

Table 1. The composition of diet fed to the crabs (% dry weight ± SD (n))

Component Bivalve Prawn 1 Prawn 2

Crude Protein(%) 3.04 ± 2.02 (3) 68.20 ± 0.07 (3) 67.20 ± 0.22 (3) (N x 6.25)

Crude lipid ( % ) 17.99 ± 0.36 (3) 13.43 ± 1.30 (3) 13.91 ± 0.17 (3)

Chitin(%) (N x 14) - 5.42 ± 0.24 (3) 6.24 ± 0.07 (3) Gross Energy (kJ.g•l)* 22.57 ± 0.08 (3) 23.03 ± 0.02 (3) 23.20 ± 0.01 (3) Ash(%) 12.66 ± 0.38 (3) 11.85 ± 0.88 (3) 12.07 ± 0.06 (3) P:E ( mg protein.kJ•1) 31.41 ± 1.01 (3) 35.37 ± 0.83 (3) 33.55 ± 0.11 (3)

* = Ash free energy; P:E = Protein energy ratio

Fat comprised 13-18% of each diet and total energy was similar in both bivalve and prawn diets at approximately 24 kJ.g-1 dry weight.

Fresh weight to dry weight conversions were made using the following relationships established experimentally for the 3 diets.

Bivalve Y = -0.043 + 0.059 X; r = 0.9269; P<0.05>0.01 28

Prawn 2 Y = 0.135 + 0.219 X; r = 0.9979; P<0.05>0.01

Y = tissues dry weight; X = wet weight

Rate of Nutrient Intake and Output

The weight specific, daily intake and output of nutrient and energy were calculated (Table 2). Intake of dry matter, protein, fat, total energy, fat energy and protein energy were similar on the three different diets. Output of dry matter, protein, fat, and total energy did not differ significantly between diets, but the loss of protein energy of prawn diet 2 was significantly greater than that for crabs fed bivalves and prawn diet 1. The loss of fat energy in the faeces of crabs fed the bivalve diet was higher than that of crabs fed either prawn diet 1 or prawn diet 2 and loss from crabs fed prawn diet 1 was significantly smaller than for those fed the prawn diet 2.

Assimilation of the Experimental Diets

The apparent assimilation coefficients (AAC) were calculated for dry matter, fat, protein and energy (Fig.2). The values of AAC for nutrients and energy were routinely high in all the diets, exceeding 90 % in all cases. The coefficients for dry matter assimilation ranged from 91.05% (bivalve diet) to 92.45%-93% (prawn diet) with the value for prawn diet 2 being significantly higher for bivalves. Protein assimilation exceeded 98% on all diets and although significant differences were found between diets these were very small (Fig.2). The coefficients of apparent assimilation of chitin were high in both diets (96%). A similar pattern was apparent for assimilation of prokin energy (Fig.3). Assimilation of fat ranged t8

2.68

0.60

0.13

17.79

12.09

0.06

0.89

1.04

± ±

Prawn

2.11

0.19

0.48

4.25 9.44

3.12

42.62

62.60

SD)

(Mean

diets

1.30

1.73

5.86

0.29

0.04

26.81 35.79

0.81

Prawn

2.98

2.23

0.49

3.85

0.18

10.09

61.62

46.15

experimental

se,rota

Scylla

7.40

0.36

0.57

0.11

2.11

11.72

± ±

Bivalve

energy

1.43

2.27

0.41

8.38

29.37

46.60

and

nutrients

)

output

)

crab.d-

and

output

crab.d-

and

crab.d-

(kJ.kg-

intake

(kJ.kg-

(gDW.kg-

Intake

(gDW.kg-

energy Daily

(gDW.kg-

energy

(gDW.kg-

energy

matter

energy

Input:

Protein

Dry

Lipid

Protein

Chitin

Fat

Total

Chitin

Table Table 2 Coot.

Output: Dry matter (gDW.kg- 1 crab.d- 1) 0.19 ± 0.03 a 0.21 ± 0.10 a 0.19 ± 0.05 a

Protein (gDW.kg- 1 crab.d- 1) 0.03 ± 0.01 a 0.26 ± 0.01 a 0.32 ± 0.ot a

Lipid (gDW.kg- 1 crab.d- 1) 0.02 ± 0.01 a 0.02 ± 0.01 a 0.02 ± 0.01 a

Chitin (gDW.kg- 1 crab.d- 1) - 0.01 ± 0.002 a 0.01 ± 0.002 a

Total energy (kJ .kg- 1 crab.d- 1) 2.03 ± 0.38 a 1.65 ± 0.92 a 1.83 ± 0.56 a

Protein energy (kJ .kg- 1 crab.d- 1) 0.02 ± 0.0la a** 0.07 ± 0.04 a** 0.30 ± 0.11 b**

Fat energy (kJ.kg- 1 crab.d- 1) 0.23 ± 0.01 a** 0.05 ± 0.02 b* * 0.17 ± 0.04 c**

Chitin energy (kJ .kg- 1 crab.d- 1) - 0.08 ± 0.03 a 0.08 ± 0.03 a

• = significant difference among diets (P<0.05>0.01) ** = significant difference among diets (P<0.05<0.01) Different superscript letters indicate significant differences between means within a row. n = 8 (bivalve and prawn diet 1) or 9 (prawn diet 2). from 94.3% on bivalve diet to approximately to 96% on prawn diets. The total energy assimilation was very similar on all three diets with an AAC of approximately 99% in each case (Fig.3). The AAC of protein and fat energy reflected the AAC values given in Fig.2.

~ 31 DISCUSSION

Dry matter

The AAC for dry matter measured in Scylla were 91.05% ± 2.37% (bivalve diet),

92.45% ± 1.33% (prawn diet 1) and 93. 78% ± 1.48% (prawn diet 2). Clearly most of the experimental diets were digestible. These values were within the range found in the lobster, Homarus americanus (65%-70% ), the prawns, Penaeus japonicus,

72.7%-76.6% and P.monodon, 97%-98% and the crayfish, Procambarus clarkii, 25%-

100% (Table 3).

From the comparative data (Table 3) it was evident that animal based diets were more suitable for omnivorous and carnivorous crustaceans, whilst starchy and plant meals were well assimilated by herbivorous crustaceans. Thus the diets fed to Scylla were excellent sources of nutrients for crabs.

Protein

The crude protein content of the experimental diet fed to Scylla was about 0.63 g protein.g·1 dry food (DF) or 63.04 % dry matter (DM)(bivalve diet), 0.68 g protein.g·1

OF or 68.20 % DM (prawn 1 diet) and 0.67 g protein.g·1 DF or 67.20 % DM (prawn

2 diet). This protein level compares well with the protein content of diets supplied to crustaceans in other studies. In juvenile blue crab, Callinectes sapidus, the protein level between 37-39 <¼- enabled good growth (Millikin, et al. 1980). In Carcinus maenas. a high level of protein (60 %) in the diet (mussel meat) enabled the fastest growth (Ade lung and Ponat, 1977). High levels of protein ( 40-60 % ) resulted in the best condition (most l'dihle meat) in adult American lobsters, Homarus americanus

(Castle and Budson. I 974). Accordingly, all the diets probably fulfilled protein 32 requirements of the crabs.

Table 3. A comparision of apparent assimilation coefficients (AAC) of dry matter from some decapod crustaceans

No. Species Diet AAC (%) Source 1 Scylla sen-ala Bivalve, prawns 91.05-93. 78 This study

2 Homarus Animal base diets 65-70 Koshio, et al. americanus (1992) 3 Penaeus japonicus Animal base diets 72.7-76.6 Koshio, et al. (1993) 4 Penaeus monodon Bivalve 97-98 Sarac, et al. ( 1993)

5 Procambarus clarkii High starch meals 81.6-100 Reich, et al (1993) Animal meals 61.3-71.6

6 P. clarkii Starch meals 63.5-88.6 Brown, et al. Animal meals 39.1-61.1 (1986)

Assimilation of protein

The coefficients of apparent assimilation for protein were high in all three diets;

98.07% on bivalve diet, 98.63% prawn diet 1 and 98.49% prawn diet 2. These for

Scylla are at the upper end of the range of values measured in other crustaceans. In the prawn, Penaeus monodon, fed bivalves range of the AAC was 97%-98% (Sarac, et al. 1993). On plant diet much lower AAC for protein were measured in the land crab, Cardi.soma guanhuni (65.9 %) and Gecarcinus laterali.s (57%). When the crabs were fed N-supplemented food (Casein, agar, glucose, sodium bicarbonate) the AAC rose to 92.6 o/c- for C.guanhuni (Wolcott and Wolcott, 1987), whilst for G.laterali.s on a soybeans-supplemented diet it reached 85.9 % (Wolcott and Wolcott, 1984).

Presumably many plant proteins arc complex and difficult to digest. Crude proteins were also found to be well digested in other crustaceans and fishes. In prawns, the

AAC of protein of Penaeus vannamei fed a practical diet (fish meal, squid meal, 33 shrimp meal, soybean meal and rice bran) ranged between 74%-90%, while the prawns fed a purified diet had a value range from 81 %-99% (Akiyama, et al. 1989).

Isonitrogeneous diets with 40 % protein level containing four different carbohydrate levels (5 %, 15 %, 25 % and 35 %) were well assimilated by Penaeus monodon and the AAC for protein ranged from 92.8 to 94.3 % (Catacutan, 1991). Artificial diets containing 40% and 44% protein levels were also assimilated efficiently by

P.monodon; 90%-94% for prawns reared in the sea water and 89%-94% when reared in brackish water (Shiau, 1991 ). Diets containing starchy seed meals and grain by products were most efficiently assimilated by Macrobrachium rosenbergii, whilst animal protein and high-fibre products had low apparent assimilation values (Reigh, et al. 1990).

The apparent assimilation coefficients (AAC) of crude protein in Scylla were very high on all three experimental diets. This is perhaps not surprising for an animal diet where much of the protein is readily available but it also suggests Scylla could digest protein from the protein/chitin complexes in exoskeletal materials of the prawn diets.

The feeding habits of Scylla se"ata, active predators of sessile and slow-moving macro-invertebrates and scavengers, probably also influenced the capability of this species to digest such diets. Clearly, the three experimental diets meet their requirements. Like, fish, the principal digestible energy of Scylla is in the form of protein (Brett and Groves, 1979).

Lipid

In the present study the crude lipid content of the experimental diet was about

0.18 g lipid.g·1 OF or 17.99 % OM in the bivalve diet, 0.13 g lipid. g· 1 OF or 13.43 %

OM on prawn diet 1 and 0.14 g lipid.g·1 OF or 13.91 % OM on prawn diet 2. The intake of crude lipid was approximately 0.41 g.kg·1 crab.d-1 on the bivalve diet, 0.49 34 g.kg crab·1.d·1 on prawn 1 diet and 0.48 g.kg· 1 crab.d·1 on prawn diet 2. The AAC of crude lipid were high in three diets; 94.3 % on the bivalve diet, 94.1.26 % on the prawn diet 1 and 95. 79 % on the prawn diet 2. Thus essentially all of the crude lipid in the diets was assimilated.

Generally speaking, the AAC of lipid observed in the present study was high compared to that reported in other decapod crustaceans. The apparent assimilation coefficient of crude lipid of the lobsters, Homarus americanus fed a practical diet ranged between 77%-90% (Koshio, 1992). Artificial diets containing dietary fat levels ranging from 8.0%-16.5% were well assimilated by Penaeus japonicus and gave the best growth and survival (Kanazawa, 1985). On the other hand, in Penaeus vannamei, the AAC of crude lipid from artificial diets were found to differ with body size.

Prawns with the weights of 22.8 g-25.2 g, 12.6 g-16.3 g and 8.4 g-11.0 g had AAC values of 41.8%-58.4%, 46.9%-53.0% and 43.9%-53.8%, respectively (Smith, et al.

1985). In Penaeus setiferus and P. aztecus, lipid from formulated diets were assimilated with lower efficiency than those from natural diets (Condrey, et al. 1972).

It is presumed that certain types of fat were more useful than others.

Unsaturated fatty acids have been found to be more digestible than saturated fatty acids in rainbow trout and mink (Austreng, 1980) and this may effect assimilation in some diets.

Energy

The intake of energy derived from protein and fat was 37.75 kJ.kg·1 crab.d-1 fed on bivalve diet, 56.24 kJ.kg·1 crab.d·1 fed on prawn diet 1 and 52.06 kJ.kg·1 crab.ct· 1 fed on prawn diet 2. Energy from other substrates was relatively small component and comprised approximately 19o/c. of the total on bivalve diet and 2.80%-10.05o/c on 35 the prawn diets. Thus only about 6.42% was derived from non protein/lipid/chitin sources and was probably carbohydrate.

In the present study, the AAC's for total energy were 99.44 ± 0.26 on the

Bivalve diet, 99.53 ± 0.16 on prawn diet 1 and 99.62 ± 0.19 on prawn diet 2. The

AAC of energy derived from protein were 97.97 ± 0.77 on the bivalve diet, 98.75 ±

0.24 on prawn diet 1 and 98.51 ± 0.43 on prawn diet 2, and from fat were 93.99 ±

1.85 on the bivalve diet, 95.75 ± 1.26 on prawn diet 1 and 96.12 ± 1.12 on prawn diet 2. Energy derived from chitin had AAC values of 95.95 ± 1.00 (prawn diet 1) and 96.15 ± 0.84 (prawn diet 2).

The AAC of artificial diets containing energy levels 19.67-21.35 kJ.g-1 dry weight were well assimilated by Homants americanus, and AAC ranged from 68-80%

(Koshio, et al. 1992). In Procambants clarkii, the AAC of energy of artificial diets contains animal material, plant and yeast were 68.1 %-82.5%, 35.6%-100% and 61.6%

(Reigh, et al. 1990).

P:E Ratio

Estimation of energy requirements is usually expressed as protein-energy ratio

(P:E ratio). The relationship between protein and energy is an important factor to measure the response of growth. In this study, P:E ratio in the diets was 31.41 ± 1.01 mg-protein.kJ•1 energy on the bivalve diet, 35.37 ± 0.83 mg-protein.kJ-1 prawn diet 1 and 33.55 ± 0.11 mg-protein.kJ•1 on prawn diet 2. The optimal levels of P:E ratio for best growth vary in crustaceans. In prawn Penaeus monodon, for instance, a diet with a P:E ratio of 32.91 mg protein.kJ•1 was found to be effective in promoting growth

(Bautista, M.N. 1986 ). In two different salinities (sea water and brackish water), the prawn, Penaeus monodon required different protein and energy levels for their best growth. Thus a diet with P:E ratio of 24.63 mg protein.kJ•1 gave highest growth rates 36 for prawns reared in sea water, whilst a P:E ratio of 27.09 mg protein.kJ•1 was best for prawns reared in brackish water (Shiau, et al. 1991 ). Best growth was observed in

Penaeus merguensis fed diets with P:E ratios ranged between 22.80-28.01 mg protein.kJ•1; this diet contained 34%-42% protein levels and 10.465 kJ.g·1 dietary energy (Sedwick, 1979). Hajra, et al. (1988) found that the optimal P:E ratio for growth was 25.97 mg protein.kJ•1 in P.monodon. In the red crayfish, Procambarus clarkii a diet with P:E ratio 28.67 mg protein.kJ•1 gave the best growth (Hubbard, et al. 1986 ). Given these values it is likely that all three experimental diets used for

Scylla more than fulfilled daily protein and energy requirements for growth. Indeed a number of crabs went into premoult before assimilation experiments were completed.

Assimilation of chitin

In this study, the AAC of chitin was high in both prawn diets; 96.40 ± 1.40 (prawn diet 1) and 96.60 ± 0. 70 (prawn diet 2). The AAC of chitin in artificial diet diets for other species ranged from 24.7 - 36.5 % (Penaeus setiferus), 31.9 - 38.9 % (P. vannamei) and 49.5 - 54.7 % (P. duorarum) (Clark, et al. 1993). In the crayfish,

Procambarus clarkii the AAC was 71.56 % (Brown, et al. 1986). On the other hand, negative apparent assimilation of chitin was observed in P. vannamei (Akiyama, et al.

1989). These researchers did not determine assimilation of chitin directly but rather inferred it from the AAC for dry matter. Chitin in the faeces was not measured.

The values for AAC of chitin from this study were considerable higher than reported for other decapod crustaceans. This may reflect the large contribution chitin makes to the diet of Scylla, a carnivore, compared to that in the other omnivorous species examined. It is also possible that fresh chitin in the uncooked prawns supplied was more digestible for Scylla than in cooked chitin. As indicated in the first trial for the diets. the crabs chose fresh chitin when fresh and cooked prawns 37 were given in the same time.

N-acetyl-D-glucosamine (NAG) has also been used as an indicator of chitin digestion in rainbow trout (Lindsay, et al. 1984) and in prawn, Penaeus monodon

(Fox, et al. 1993). The inclusion of glucosamine in the diets has been also found to

be a growth-promoting factor (Kitabayasi, et al. 1992 in Fox, et al. 1993).

In conclusion, Scylla was fed ad libitum in these experiments and as some food

always remained uneaten after each feeding period the feeding regimes clearly

provided adequate food. In support of this, many of the experimental crabs moulted

under the holding conditions suggesting the diet was sufficient to allow growth.

The sum of protein and fat in the experimental diet was lower than that of total

nutrients and the balance must have been made up by other nutrients, presumably

largely carbohydrate. Chitin was well assimilated in Scylla as in other decapod

crustaceans. Values for the assimilation of energy suggest 6.42% of this may have

been derived from chitin from the exoskeleton in prawn diets or other sources.

From the assimilation point of view, it appeared that Scylla may be able to utilise

these natural diets very efficiently but their effect on growth would need to be

confirmed in growth trials. All of the assimilation values in the present study were

higher than reported in assimilation studies in the literature. This was probably

because the diets used were natural diets, well suited for efficient anabolic utilization

of nutrients by the carnivorous Scylla. Both diets, however are expensive and for

aquaculture it would be necessary to establish an artificial diet which was cheaper but

still suitable for Scylla. Protein is the most expensive ingredient in diet formulation

and the protein content could be reduced so that it was not used as an energy

source. It could be substituted with other nutrients (such as lipid and chitin) to

supply the necessary energy whilst maintaining protein content at a level adequate for

growth. Consequently. it is necessary to known the optimal protein, fat and chitin 38 levels in the diet for the best growth of Scylla. 39 RATE OF PASSAGE STUDY

3.1. INTRODUCTION

The speed of movement of food through the alimentary tract has been expressed in numerous different ways in the literature. The most useful definitions are the rate of passage, the rate of flow of digesta and the rate of digestion. The rate of passage is the length of time required for undigested materials from a given meal to be eliminated in the faeces. The rate offlow of digesta is the length of time required by the mixture of undigested materials from previous meals to be eliminated in the faeces (Balch, 1961 ). The rate of digestion is the total time needed for the foregut to empty after a given meal. Other terminology used in the literature includes "gastric evacuation rate","gastric digestion rate", "gastric emptying rate", "gastric elimination rate", "foregut evacuation rate" and "foregut clearance rate" (Fange and Grove, 1979).

As these "rate" definitions are not true rates, but represent time only, MRT is preferred in this study.

Several methods have been used to study the rate of passage of food in the digestive tract of animals including radioisotopes and an x-ray method (Molnar, et al.

1967; Edwards, 1971). Both in domestic and aquatic animals, radioisotopes have been commonly used as indigestible markers (Downes and McDonald, 1964; Windell,

1968). In domestic animals, indigestible markers which have been used include the particulate markers Cerium-144, Praseodymium-144 and 103 Ru-phenanthroline, and the soluble markers for fluid digesta Chromium-51 with EDTA and

Polycthylcneglycol (Grovum and Williams, 1973; Faichney and Griffiths, 1978).

Polyethyleneglycol (PEG) which is the most extensively used as a water soluble marker, has an average molecular weight of about 4000, and is not absorbed in the gut of ruminants (Hyden, 1960). 40

In aquatic animals, 137·cesium was used for determination of the evacuation rate of bluegill, Lepomis macrochirus (Kolehmainen, 1974) and 51 -chromium was utilized in determination of assimilation efficiencies (Calow and Fletcher, 1972; Wightman,

1974 ). Studies on the rate of passage in food of crustaceans have been quite limited until recently. Foregut evacuation rate was studied in lobsters, Nephrops norvegicus

( Sarda and Valladares, 1990), in Jasus lalandii (Pollock, 1979) and in Panulirus longipes (Dall, 1975). The commonest method used was to kill animals at various times after feeding, but this requires a lot of experimental animals to be sacrificed and is therefore better suited to small rather than large species. The present study was designed to avoid killing large number of (expensive) crabs and used markers to determine the rate of passage of digesta. Three indigestible markers were tested; st

Cr EDTA and PEG were investigated as markers for the soluble food fraction and 57-

Co labelled microspheres were used to mark the particulate fraction.

3.2. MATERIALS AND METHODS

3.2.1. Particle Markers

3.2.1.1. Experimental apparatus

The crabs were maintained in a temperature and humidity controlled room as detailed previously (Chapter 2. General materials and Methods)

3.2.1.2. Experimental animals

Eleven crabs (381-686 g and carapace length 8.2-9.5 cm) were used. Six of them were used in the study with particulate markers and the others were used for the solute markers.

3.2.1.3. Dietary treatment for particle and soluble marker experiments. 41

The crabs were fed with fish and acclimated to the experimental conditions and experimental diets for 7 d. The food was stored frozen, but thawed before being given to the crabs. Food was normally supplied at 1800 h and given ad libitum. The crabs were fasted 1 d prior to injection with markers. They were fed immediately after the marker was injected thereafter food was supplied daily at 1800 h as usual.

3.2.1.4. Marker used

Radioactive microspheres <51·co, 15 µm, 542.8 MBq g·1) were used as a particulate marker and provided by NEN-TRAC microspheres Dupont. Each crab was given a measured dose containing approximately 3.3 MBq (3.3 x 106 DPS) in sea water. The experimental crab was strapped to a board ventral side uppermost and the marker was injected into the foregut through a catheter tube inserted via the mouth. They were immediately put into the experimental tank and fed ad libitum.

3.2.1.5. Collection and storage of sample

A plastic bag was attached to the abdomen as detailed previously (Chapter 2).

Faecal samples were collected from the plastic bag using two catheter tubes to flush the bag. For this purpose, the crab was again secured to a board ventral side uppermost and the faeces were sucked out with a syringe. Faeces for individual crabs were collected daily and kept individually in plastic vials until measurement of radioactivity. The collection of faeces was initially planned at two hour intervals.

However, the crabs generally defecated only once or twice a day, usually late at night or early in the morning, and sampling was therefore conducted each morning.

3.2.1.6. Radioisotope counting

Radioactivity was measured using a modular Scintillation counting system with 42

ORTEC and TENNELEC modules and a BICRON well scintillation detector.

Samples were made up to 3 mL in plastic tubes and counted until 10,000 counts were recorded. Sample counts were corrected for background radiation.

3.2.2. Soluble markers

3.2.2.1. Markers used

In a preliminary study, 51 -cr EDTA was tested as a soluble marker. However, absorption occurred over the gut and 51 -cr appeared in the blood. 51 -cr EDTA, therefore, was not a suitable marker for Scylla se"ata; a prerequisite for a gut marker is that it not be absorbed. A similar capacity to absorb 51-Cr from 51-Cr EDTA in the gut was reported for Birgus latro (Greenaway, et al. 1990).

Polyethylene glycol 4000 (BDH Limited Poole England, average mol wt, 3600-

4400) was then tested as a marker for the fluid fraction of the digesta. PEG was not absorbed in the gut of sheep, swine or chickens, Gallus domesticus (Hyden, 1961;

Ishikawa, 1964; Bindslev and Skadhauge, 1971) and was considered to be a satisfactory marker in these species. Tests were conducted to determine whether

PEG was absorbed across the gut or whether it was confined in the lumen in Scylla.

Midgut gland tissues and blood were analyzed for PEG following its introduction into the gut. The results showed that PEG was not detectable either in the midgut gland tissue or in blood. Thus PEG was considered to be a satisfactory marker for use in

Scylla. Each crab was given 2 mL of 10 mg.mL1 PEG injected into the foregut via a cannula and the crab was treated as described above (section 3.2.1.3).

3.2.2.2. Collection of samples

To remove the faeces collected in plastic pouches, the pouch was opened and solid materials were removed with forceps, whilst the watery materials were collected 43 by suction with a syringe. Faeces from each individual crab were collected daily and kept individually until analyzed.

3.2.2.3. Sample analysis

PEG was determined using Malawer and Powell's method (1967). Samples consisted of faecal material from the plastic bag, digestive juice from foregut and midgut gland tissues. Preparation of ZnSO4 and Ba(OH)2 was performed as suggested by Annino (1964). Assay mixtures for PEG determination (Table 4) were prepared in the glass tubes.

Samples, water, BaC12, Ba(OH)z were added, respectively and mixed after each

addition. ZnSO4 was then added and the mixture was shaken vigorously. The mixture was filtered through Whatman no.42 filter paper. To 1 mL filtrate was added 3.0 mL of gum arabic (Ajax chemicals, 12.0 mg.L-1) and the resultant was mixed gently.

Finally, the mixture and 4 mL of trichloroacetic acid (TCA) containing 0.20 mol.L1

BaCl2 anhydrous were blended. The optical densities of samples were read after 60 to 90 min using an LKB Ultraspec II

Table 4. Assay mixtures for PEG determination

No Solution Volume mL 1 Sample 1

2 Water 10

3 BaCl1 (0.41 mol.L- 1) 1

4 Ba(OH)~ (0.15 mol.L1) 2

5 ZnSO. (0.17 mol.L 2 Total 16 44

Standards were prepared as following concentrations: 200, 400, 600, 800 and 1000 mg.100 mL- 1 PEG; In standard tubes, the sample was substituted with an equal volume of standard solutions; In blank tube, the sample was substituted with an equal volume of water. Tubes were read on a spectrophotometer (LKB Biochrom,

England) at 410 nm. A blank and PEG standards were run with each batch.

3.2.2.4. Data analysis

The excretion of both markers was expressed as a cumulative percentage of total marker excreted in the faeces. The times needed for the excretion of 5 %, 50 % and

95 % of the total marker eliminated were calculated on the assumption that faecal production throughout the collection period was constant. The mean retention time

(MRT) was calculated by adding together the times of 5 % to 95 % at 10 % intervals and dividing the sum by 10 (Castle, 1956; Herd and Dawson, 1986).

3.2.2.5. Statistical analyses

The Student's t-test was used to compare means using the independent test in the Systat Software Package (Systat,Inc.1990). All percentage data were transformed to arcsin values prior to analysis using QUATTRO PRO Software Package

(Borland,lnc.1990). The criterion for significance was set at P<0.05 unless otherwise stated. 45 RESULTS

Particle marker

In the present study, the particulate marker first appeared in the faeces 20.33 h after dosing. The mean value for total particle marker excreted in the faeces during the experiments (308.17 h) was 86.66 % ± 7.99 %. Excretion of particle marker 5%,

50% and 95% of the total marker eliminated was showed (Table 2). The MRT of particle marker was 19.16 h. ± 6.89 h.

Table 5. Cumulative particle marker excretion in Scylla serrata

Excretion Time (h) (n=6) 57•co

(%) Mean ± SD 5 1.92 ± 0.69 50 19.16 ± 6.89 95 36.39 ± 13.10 MRT 19.16 ± 6.89

Fluid marker

Excretion of the fluid marker PEG was very slow and increments were recorded daily through the experimental period of 648 hours. At the end of this period only

12.51 ± 5.95 o/c of the injected dose of PEG had been released in faeces. To o..t determine the amount emerging in the gut @ this time a catheter was inserted and as much fluid as possible was withdrawn from the foregut and analyzed for PEG. The mean PEG content of the juice accounted for a further 46.01 % ± 6.69 % of injected 46 dose. As considerable fluid must have remained in the midgut gland tubules it was likely that most of the remaining 41 % of injected PEG also remained in the gut. 47 DISCUSSION

Particle markers

Histochemical studies showed that foregut digestion in Scylla was nearly complete

12 h after feeding (Barker and Gibson, 1978). In a second investigation, slaughter experiments showed that the foregut digestion for soft tissues was complete about 12 h after feeding but that large shell and bone required 5-6 d (Hill, 1976). A study of gastric digestion in the rock lobster, Panulirus cygnus using two species of algae, the gastropod, Littorina unifasciata and foot muscle of the abalone, Haliotis roei as foods showed a faster digestion time of 4-6 h (Joli, 1982). The above the studies suggested that undigested particles from a meal should largely reach the faeces within 12 h after eating but would continue to arrive for several days where there are significant bone or shell components.

The mean retention time of 19.16 h in Scylla was long considering the much shorter time apparently needed for digestion. Scylla is a carnivore and shows very high assimilation of the diet (see Chapter 2). A small amount of faeces (particulate matter) was therefore produced and generally voided only once a day. Thus, despite a relatively rapid digestion rate (approximately 12 h) the earliest time the particle marker could appear in the faeces was about a day after its injection and this limited the accuracy of the method. More accurate results are obtained with animals which produce numerous faecal pellets at frequent intervals.

The MRT in Scylla was longer than in the bird, Dromaius novaehollandae, but it was faster than in the goat and the tammar fed one meal per day and it was the same as in tammar wallaby fed continuously. This situation was presumably a reflection of the feeding habits of the crabs. They have non continuous feeding habits eating a lot of food early in the evening and a smaller amount late at night or 48

early in the morning (Hill, 1976). There is an evidence that the tammar wallaby,

Macropus eugenii, fed one meal per day had a MRT longer than that measured

during continuous feeding (Warner, 1981).

Table 6. Comparison of cumulative marker excretion in Scylla serrata and other species

Marker/Species MRT Source (h)

A. Particulate marker l. Scylla serrata 19.16 ± 6.89 This study 2. Emu, Dromaius 5.5 ± 0.4 Herd and Dawson (1984) novaehollandae 3. Goat 38.0 ± 3.6 Castle (1956) 4. Tammar wallaby, 29.3 OMPD Warner (1981) Macropus eugenii 19.1 CF

B. Fluid marker

1. Scylla serrata 133.95 ± 73.11 This study 2. Emu, Dromaius 4.1 ± 0.2 Herd and Dawson (1984) novaehollandae 15.7 OMPD Warner (1981) 3. Tammar wallaby, 11.3 CF Macropus eugenii

MRT= mean retention time in h; OMPD= one meal per day; CF= continuous feeding; Means± SD

The data presented above indicated that 57·Co moved freely through the digestive

tract of Scylla and was satisfactory a marker for particles. Use of Radio-microspheres

had the important advantage that the analytical procedure was quite precise. Samples

were readily counted in a scintillation counter and the high energy gamma radiation

allowed simple preparation of the specimens for analyses.

Fluid marker

Only about 12.51 o/t- of PEG injected into the gut was subsequently collected in 49 the faeces over the time period of experiment (648 h). The marker, however, was not detected in either blood or digestive gland tissues and this low recovery could not be attributed to absorption from the gut. PEG seemed to be a satisfactory marker in this respect and to behave much as reported in mammals. It seems likely, therefore, that PEG was being retained in the gut and indeed this was confirmed by analyses of digestive juice at the end of the experiment which indicated large amounts of PEG to be present in the gut lumen. In crustaceans, digestive juice is secreted by the midgut gland and passed forward to the foregut. Digestive products and fluid are then reabsorbed in the midgut gland. Presumably PEG is recirculated between midgut gland and foregut with only small losses occurring to the hindgut to be eliminated with the faeces. As it is not digested or absorbed, PEG will remain in the gut for a long period. Thus whilst PEG fills the criteria for an indigestible fluid marker, the concept of linear movement of fluid fraction developed for vertebrates with linear guts is inapplicable to the 'recirculatory' digestive system of crabs. It was not possible to calculated MRT values as only a small amount of the total PEG injected was excreted and PEG continued to appear in the faeces at low but steady rate even after

648 h. 50 ENZYMATIC STUDY

4.1. INTRODUCTION

Major organic components in organisms are the polymer cellulose, chitin and collagen. Chitin is structurally related to cellulose, but is more complex than either cellulose or collagen. Its structure is shown in Fig. 1. Chitin contains less than 7 % nitrogen and is commonly associated with proteins, lipid and inorganic salts, e.g. calcium carbonate. It called chitosan if the nitrogen content is more than 7 %

(Tracey, 1955; Hackman, 1971; Stevenson, 1985; Davies and Hayes, 1988; Sandford,

1989).

Structurally, chitin is a polysaccharide consisting of unbroken chains of N-acetyl

D-glucosamine and it is an important structural component not only in the animal but also in the plant kingdoms. In nature, chitin is not a pure compound, but always occurs as a chitin-protein complex (Hackman, 1960; 1962). In crustaceans and insects it is a major structural component of the exoskeleton and is synthesized and degraded many times during their life time as part of the moulting cycles (Hackman,

1970; Neville, 1975). Using X-ray diffraction; three polymorphic forms of chitin a,{3 and y differing in their crystal structures, can be distinguished (Rudall, 1963). a chitin occurs in larvae of honeybees, locusts, cockroaches and the beak of squid, whilst {3-chitin is found in the chaetae of annelids and the pen of squids. y-chitin occurs in the gastric cover of Loligo, the peritrophic membranes of dragonflies, locusts, cockroaches and larvae of silk worm (Hackman,1960; Brimacombe and

Webber. 1964; Muzzarelli. 1976; Peters, 1992).

Three enzymes are involved in the degradation of chitin. Firstly, it can be cleaved by chitinase (chitin glycanohydrolase EC 3.2.1.14) to yield oligomeric, trimeric and dimeric sugars (chitobiose, chitotriose). The second enzyme, {3-N- 51 acetylglucosaminidase ( chitobiose acetylaminodeoxyglycohydrolase EC 3.2.1.30;

NAGase) breaks down the dimer ( chitobiose) to N-acetylglucosamine. Lysozymes

(EC 3.2.1.17) also hydrolyse chitin at a slow rate (Clark et al. 1988; Zikakis and

Castle, 1988).

Chitinases can be classified as endo- and exo-type enzymes based on the initial reaction product formed (Koga, et al.1982; Koga, et al. 1986; Bade and Hickey,

1989). Thus, endochitinase [endo-/3-(1-4 )-poly-N- acetylglucosaminidase; chitinase EC

3.2.1.14] breaks down internal linkages between units of chitin producing oligomers of 2 or more subunits (Fig. 2). Endochitinase from the midgut gland of the prawn,

Penaeus japonicus reduces pentamers to dimers and trimers (Kono, et al. 1990).

Wheatgerm also splits chitin into oligomers (Cabib, 1988), whilst endochitinase from the fungus, Pycnoporus cinnabarinus (Otahkara, 1988) reduces oligomers to dimers.

Endochitinases have also been found in the leaves of the bean, Phaseolus vulgaris

(Abeles, et al. 1970; Boller and Vogelli, 1984) and in the tomato, Lycorpersicon esculentum (Pegg, 1988).

Exochitinase [exo-/3-( 1-4 )-oligo-N-acetylglucosaminidase; /3-N acetylglucosaminidase; EC 3.2.1.30; (NAGase)] splits the non-reducing end of 2- acetamido-2-deoxy-/3-glucose residues in dimers but is also reported to break down trimers and tetramers (Jones and Kosman, 1980; Zikakis and Castle, 1988).

Exochitinases have been detected in the crab, Cancer borealis and C. irroratus (Brun and Wojtowil-'Z, 1976), in the lobster, Homarus americanus (Lynn, 1990) and in the two euphausids, Euphausia superba and Meganyctiphanes norvegica (Spindler and

Buchholz, 1988), in the lymphomyeloid tissues of marine fishes (Lundblad, et al.

1979), in Bombyx mori (Kimura, 1976), in the digestive fluid of spider, Cupiennius sa/ei (Mommscn, 1980) and in the sawtoothed grain beetle, Oryzaephilus surinamensis

(Fukamizo. et al. 1985). Exochitinascs from Serratia marcescens, Pseudomonas stutzeri 52 and Streptomyces griseus were capable of hydrolysing chromogenic substrates, p nitrophenyl-/3-D-N,N' -diacetylchitobiose (Roberts and Selitrennikoff, 1988). S. plicatus also has the ability to break down analogs of trimers and tetramer derivatives

(Robbins, et al. 1988).

These chitinolytic enzymes are synthesized in, and secreted by, the pancreas of vertebrates, the gastric mucosa of amphibians and reptiles (Jeuniaux, 1961; 1966), the gastric or pancreatic gland cells of fish (Fange and Grove, 1979) and the midgut glands of crustaceans (Barker and Gibson, 1979). The enzymes may have different physiological functions in different organisms; in the higher plants, for example, they function in defence against arthropod pathogens (Bohler, 1988; Roby,et al. 1988). In animals, the enzymes are involved in the moulting process of insects (Kimura, 1973;

Bade,1974), crustaceans and in hatching of eggs in brine shrimp (Artemia salina) and probably other crustaceans. They also function as digestive enzymes in organisms ranging from Protozoa to mammals (Spindler, 1983) where chitin occurs in the diet.

The chitinolytic enzymes in the digestive gland of crustaceans have been investigated in a number of species; Cancer borealis and C.irroratus (Brun and

Wojtowicz, 1976), Penaeus japonicus (Koga,et al. 1990; Kono,et al. 1990) and

P .setiferns (Lee and Lawrence, 1985). Chitin degrading enzymes have also been examined in the cysts of brine shrimp, Artemia (Funke and Spindler, 1989) and in the integument and digestive tract of Antartic krill, Euphausia superba (Buchholz, 1989) and Palaemon serratus (Spindler-Barth, et al. 1990). In the latter, activity of chitinase and /3-N-acetylglucosaminidase (NAGase) in the digestive gland were high during the moulting t--ycles. The activity of both enzymes in the integument was low during newly and postmoult and intermoult stages and increased during premoult stages. Few studies have investigated the activity of the enzymes or their characteristics in digestive juice of decapods. A study of chitinolytic enzymes in the foregut juice of 53

Homarus americanus (Brokerhoff et al. 1970) identified NAGase, but not chitinase activity. On the other hand, Lynn (1990) found activity of both enzymes in the foregut juice of the sample species. Chitinase activity has also been observed in the foregut juice of Astacus fluviatilis (Kooiman, 1963).

The present study was designed to examine the properties of chitinolytic enzymes in the foregut juice and midgut gland of Scylla serrata.

4.2. MATERIALS AND METHODS

4.2.1. Experimental apparatus

Crabs were maintained in a recirculatory aquarium as described earlier (General

Methods chapter 2).

4.2.2. Dietary treatment

Experimental crabs were fed ad libitum on prawns. A prawn diet was selected to ensure substantial amounts of chitin were present in the diet. The food was normally supplied daily at 1800 h, but during enzymatic studies the time of feeding was changed to 0800 h, to enable assay procedures to be completed during working hours. The crabs were acclimated to this regime for 5 d.

4.2.3. Preparation of substrate for chitinase assay

Colloidal chitin was prepared by modifications of Boden's method (1985). Crab chitin from Sigma (50 g) was boiled at 100°C for 2 h in 600 mL of 2 mol.L-1 KOH, then filtered with Whatman paper no.1 and washed with deionised water. The remaining chitin was then placed in 500 mL of formic acid (26.42 mol.L- 1) at 4°C with continuous stirring. This solution was filtered and washed with deionised water.

Next, the chitin was dissolved in 600 mL cone. HCl and stirred for 12 h. The mixture was filtered through glasswool. The filtrate was stirred continuously at 4°C and 54 precipitated by adding water. The supernatant was decanted and the remaining colloidal chitin was washed with distilled water until the filtrate reached neutral pH.

The colloidal chitin was washed with ethyl alcohol, acetone and finally with diethyl ether. It was suspended in a 0.2 mol.L1 Mcilvaine buffer (pH 5.5) and sonicated

(Branson-Sonifier Cell Disruptor, Branson Ultrasonic Corp. USA) until the colour changed from whitish-yellow to clear-white. The chitin was finally stored in a freezer at a concentration of 50 mg.mL-1 in this buffer.

4.2.4. Buffers for enzyme assays

Two universal buffers were used in the enzymatic assays. Citrate-phosphate

(Mcilvaine) buffer covering a pH range from 2.2 to 8.0 was employed for most assays

(Mcilvaine, 1921 ), whilst the Britton-Robinson buffer (pH range 2.6 to 11.8) was utilised to study the effect of pH on the enzymes activities (Britton and Welford,

1937).

4.2.S. Sample collection and preparation for NAGase and chitinase assays.

Collection of foregut juices

To collect juice from the foregut, the crab was strapped to a board, ventral side uppermost, and a catheter tube was inserted into the foregut via the mouth. The juice flowed along the catheter by gravity and was collected in a closed Eppendorf tube and stored for a short period ( <30min) on ice. The pH of the juice was measured using a Radiometer capillary electrode (G297) in a Radiometer BMS 3 Mk

2 Blood Microsystem thermostatted to 25°C and an Acid-Base Analyzer Model PHM

71 (Radiometer-Copenhagen). 55

Preparation of foregut juice

Foregut juice was taken from the ice-box and diluted 1: 1 with 0.2 mol.L- 1

Citrate-phosphate buffer pH 7 or in 0.1 mmol.L-1 Britton-Robinson buffer in the pH range 4-9. The juice was then centrifuged (Biofuge 17RS Heraeus Sepatech) @

10,000 g for 20 min at 4°C. The supernatant was employed for the enzymatic activities.

Preparation of digestive gland extracts

Experimental crabs were killed by chilling in a freezer. The digestive gland tissue was immediately excised and a sample was taken and diluted in Mcilvaine buffer, pH

7, containing 0.2 mmol.L-1 phenylmethylsulfonylfluoride and 0.1 mol.L-1 dithiothreitol

(DTT) at a ratio of diluent to sample 1.5:1. It was then homogenized with a tissue grinder (Tekmar, SDT Tissumizer) at 18000 rpm for 5 seconds. The homogenate was centrifuged @ 10000 g for 20 min at 4°C and the supernatant was used for the enzyme assays and for protein determination. The effect of including the protease inhibitors PMSF and DTT on activity of the enzymes was measured. The result showed that the inclusion of protease inhibitors had no significant effect on the activity of chitinase or NAGase (P=0.58 and P=0.13, respectively). The protein in the supernatant was determined by the protein-dye binding method of Bradford

(1976) employing bovine plasma gamma globulin as a standard, described by Bio-Rad

Laboratories, Richmond, CA.

4.2.6. NAGase assay

p-Nitrophenyl-N-acetyl-/3-D- glucosaminide (p-NAG, Sigma) was used as a substrate and liberation of p-nitrophenol from p-NAG was used as a quantitative indicator of N-acetyl-/3-D-glucosaminidase activity. This method has been widely 56

employed (Boden et al. 1985; Spindler and Buchholz, 1988; Lynn, 1990) and allows sensitive determination of V max• K,,, and K; values for N-acetyl-/3-glucosaminidase in

both crude and purified preparations (Boroah, et al. 1961; Woollen, et al. 1961).

In this study, NAGase assay mixtures were prepared in semi-micro disposable

cuvettes (Plastibrand) according to the outline in Table 1. The mixtures were then

incubated at controlled temperature in an LKB Ultrospec II spectrophotometer

(LKB Biochrom, England) at 25°C and read @ 410 nm. The temperature was

checked with copper/constantan thermocouple and microvoltmeter (HR 33T Wescor

Inc., Logan, Utah) before the enzyme was applied. Reaction velocity (production of

p-nitrophenol) was monitored with an IBM compatible PC using an LKB Enzyme

Kinetics Software Package. Activity was expressed as µmol.min- 1.mL1 enzyme solution

or µg.min- 1 .mg-• protein for midgut gland homogenates (modified after Spindler,

1976).

Preliminary trials showed that the NAGase activity decreased when sea water was

included in the assay mixtures and as the inorganic composition of digestive juice is

similar to that of sea water it was necessary to measure enzyme activity under similar

condition to enable approximation of in vivo activity. Thus the diluent used (Table 1)

had a composition appropriate to the measure ionic composition of foregut juice and

contained 529 mmol.L-1 NaCl, 13.36 mmol.L-1 KCl, 15.95 mmol.L-1 CaCl2, 24.35

mmol.L-1 MgCl1. 57

Table 7. Assay mixtures for determining NAGase activity

Solution Volume µL p-Nag 5

Buffer 130

Diluent 600

Enzyme 15 Total 750

In control tubes, the volume of p-NAG was substituted with an equal volume of buffer.

Effect of pH on absorbtion at 410 nm by p-Nitrophenol

Preliminary trials indicated that the absorption @

410 nm by p-Nitrophenol (PNP) was pH dependent (Fig. 7). Trials were therefore conducted to determine if the pH of the reaction mixture altered during activity measurement. Results showed pH of the mixture before and after the reaction did not differ significantly (P=0. 71 ). Calibration curves for PNP @ each assay pH were made and used to determine the appropriate extinction coefficient for activity measurement at different pH values. A typical assay curve for NAGase is shown in

Fig.8.

4.2. 7. Chitinase assay

A modification of the procedure of Boden,et al. (1985) was used to determine levels of chitinase activity in foregut fluid and digestive gland tissue. This method has also been used successfully by Spindler and Buchholz (1988). Chitinase assay mixtures (Table 2) were prepared in stoppered Eppendorf tubes. The mixtures were 58

incubated with continuous stirring at 35°C for 20 h.

Table 8. A<;say mixtures for determining chitinase activity

Solution Volume µL Colloidal chitin 10

Buffer 140

Enzyme 25 Total 175

In control tubes, the colloidal chitin was substituted with an equal volume of buffer.

After incubation, the mixtures were centrifuged (Biofuge 17RS Heraeus Sepatech) @

10,000 g at 4°C for 10 min. The supernatant (100 µL) was then incubated with 20 µL

{3-glucosidase (5mg.mL·1, Sigma) for 2 h at 35°C. At the end of the incubation, the

reaction was stopped by addition of 100 µL borate buffer (1.12 mol.L·1 boric acid and

0.56 mol.L1 KOH, pH 9.2, prepared fresh daily) and 20 µL acetic anhydride (10.58

mol.L1 in acetone, prepared fresh daily) and heated in closed Eppendorf tubes for 6

min at 95°C, then cooled in a freezer for 30 min. In the cold condition 710 µL p

dimethylaminohenzaldehyde (0.09 mol.L·1 p-dimethylaminobenzaldehyde in glacial

acetic acid, containing 0.02 mL of 31.82 mol.L-1 HCl, prepared fresh daily) and 50

µL ethanediol were added. The mixture was incubated at room temperature for 3.5 h.

The absorhance was read at 585 nm.

4.2.9. Calculation of K111 and V.,, ..

~ and V max were calculated using the direct linear plot method (Eisenthal and

Cornish-Bowden. 1974; Fig.5). This method was preferred as it avoids the problems

of unc4ual distribution of errors inherent in reciprocal plots, such as Lineweaver-

Burk plots (Henderson. 1992). Additionally. Atkin and Nimmo (1975) tested seven 59 different methods of estimation ~ and V max and found that the direct linear plot was the most satisfactory.

4.2.9. Statistical analyses

All percentage data were transformed to arcsin values prior to analysis using

QUATIRO PRO Software Package (Borland,lnc.1990). The Student's t-test was used to compare means using the independent test in the Systat Software Package

(Systat, Inc. 1990). The criterion for significance was set at P<0.05 unless otherwise noted. 60 RESULTS

4.3.1. The pH of the foregut juice

The pH of foregut juice of non-feeding crabs ( either fasting crabs or fed crabs sampled just prior to their daily feed) was significantly lower than that of crabs which had been fed (Table 9). After three days without food, foregut juice of fasting crabs was lighter in colour and more yellowish than that of fed crabs.

4.3.2. The effect of feeding and fasting on chitinolytic activity of foregut fluid

NAGase activity in foregut juice of crabs, taken before and after feeding was assayed @ pH 7 (Fig.11 ). The activity of the enzyme did not differ significantly between the two conditions. Chitinase assays exhibited similar results; the activity of foregut enzyme in digestive juice before and after feeding did not differ significantly

(Fig.12).

NAGase activity was also measured in fed and fasting crabs (Fig.13). Again there was no significant difference between the activities of enzymes from the two groups of crabs, at pH 7. A similar result was also obtained for chitinase activity (Fig.14); fasting and feeding had no effect on the enzyme activity at pH 7.

4.3.4. Kinetic properties of chitinolytic enzymes

A direct graphical evaluation of the characteristic kinetic parameters (K,., and

V max) for the enzyme, at pH 7, was made using the Direct linear plot method (Fig.10;

Table 10). NAGase has an apparent K,., value of 0.11 ± 0.06 mmol.L1, with Vmax

2.5.75 ± 36.84 µmol.min· 1.mL1 digestive juice. Chitinase has an apparent Km value of

4.32 ± 2.65 mg.mL1. with Ymax 34.85 ± 43.74 µg.h· 1.mL· 1 digestive juice. 61

Table 9. The pH and properties of the foregut juice

Crab Before After Fasting Physical Physical # feeding feeding properties properties pH pH pH before and fasting after crab feeding

1 5.9 6.7 5.8 brown, yellow- foaming brown, foaming 2 5.9 6.5 5.9 brown, yellow- light foaming brown foaming 3 5.5 6.2 5.7 brown, yellow-light foaming brown foaming 4 5.9 6.5 5.6 brown, yellow-light foaming brown foaming 5 5.8 6.9 5.9 brown, yellow-light foaming brown foaming 6 5.9 6.5 5.7 brown, yellow-light foaming brown foaming

5.8 a 6.6 b 5.8 a Mean 0.2 0.2 0.1 SD

Different superscripts on mean values indicate significant differences between means p<0.05 62

Table 10. Kinetic properties of the chitinolytic enzymes Scylla sen-ala

Substrate Concentration ~ vma't Enzyme range p-NAG Chitobiase 0.035 - 0. 701 0.11 ± 0.06 40.10 ± 41.67

Colloidal chitin Chitinase 0.5 - 4.0 4.32 ± 2.65 34.85 ± 43. 74

Values of ~ and V max were calculated using the direct linear method (Eisenthal and Cornish-Bowden, 1974); n= 6; For chitobiase ~ values are expressed as mmot.L·1 and V max values are expressed as µmol.min• 1 .mL·1 digestive juice for p-NAG substrates; For chitinase ~ values are expressed as mg.L·1 and V max values as µg.h· 1.mL1 digestive juice. Concentration was mmot.L·1 for p-NAG substrate and mg.mL1 for colloidal chitin substrate.

4.3.4. The effect of pH on enzyme activity

The effect of different pH values (range 4-9) on the rate of hydrolysis of p-NAG substrate by NAGase from foregut fluid of fed crabs was examined (Fig.15). Values are presented as percentage of the highest activity value in the pH range used. The results indicated two peaks of activity; a small peak at pH 4 and a much larger peak at pH 8.

Chitinase activity of the foregut fluid of Scylla was also affected by pH. Activity was low at pH 4 to 5 but it rose steadily from pH 6 to the pH optimum 8 (Fig.16). 63

Table 11. Specific activity of chitobiase and chitinase in the digestive gland of Scylla serrata Forskil

Chitobiase Chitinase Crab# Mean ± SD Mean ± SD 1 9.22 ± 0.23 0.17 ± 0.01

2 7.60 ± 0.36 0.16 ± 0.01

3 8.79 ± 0.31 0.15 ± 0.01

4 9.22 ± 0.23 0.16 ± 0.01

5 88.85 ± 4.65 0.33 ± 0.03

6 10.07 ± 0.69 0.24 ± 0.03

7 13.58 ± 0.16 0.31 ± 0.02

8 11.46 ± 0.20 0.28 ± 0.01 Mean± SD 19.85 ± 27.94 0.22 ± 0.02

Chitobiase; Replicates = 4; Activity was expressed as µmol.min• 1.mg·1 protein; Temperature 25°C. Chitinase; Replicates = 4; Activity was expressed as µg.min• 1.mg·1 protein; Temperature 25°C.

4.3.6. The effect of temperature on enzyme activity

NAGasc activity (at pH 7) was measured at 15, 20, 25, and 30°C covering the

normal range of water temperature experienced by the crabs. Activity increased

exponentially over the temperature range examined (Fig.17).

Chitinase activity showed a similar pattern, increasing rapidly over this range with

the highest activity at 30°C, at pH 7 (Fig.18). 64

4.3. 7. Activity of cbitinolytic enzymes in the midgut gland

The specific activities of J3-N-acetylglucosaminidase and chitinase in the midgut gland of Scylla fed on prawns were measured (Table 11 ). The activity of NAGase was highly variable between individual crabs ranging from 7.60 to 88 µ mol.min•1.mg- 1 protein. Chitinase specific activity also varied between 0.16 - 0.31 µ g.min-1.mg- 1 protein in each individual crabs. 65 4.4. DISCUSSION

4.4.1. pH of digestive juice

In the present study, a pH value ranging from 5.5 to 6.8 was found for the forcgut fluid of Scylla and there was a significant difference in pH between fasting and feeding crabs. The pH value was higher in recently fed crabs, a feature also shown in other crabs e.g. in fasting Vea marionis and U. annulipes the pH of digestive juice was 7.0-7.1, and this value increased to 7.4-7.6 after feeding (Altevogt, 1957 cited by Gibson and Barker, 1979). In starved crayfish, (Astacus jluviatilis) the pH of juice was about 5.1, but this increased to 5.6 in feeding animals (Vonk,1960). The pH of foregut fluid in the freshwater crayfish, Paranephrops zealandicus was 5.5 ± 0.3

(Musgrove, 1988) and pH 5 in the lobster, Homarus americanus (Brokerhoff, et al.

1970). Thus the pH of digestive fluid of Scylla was within the range measured in other decapod crustaceans.

4.4.2. Feeding and fasting crabs

In Crustacea, the midgut gland is the principal site of biosynthesis and degradation of nutrients and of digestive juice secretion. Additionally it functions as a store of various organic and inorganic materials used during moulting (Passano,

1960). The utilization of metabolic reserves varies considerably among groups of invertebrates (Giese, 1966). In the crab Carcinus maenas, for example, carbohydrate in midgut gland was utilized in preference to lipid or protein during starvation

(Heath and Barnes, 1970). Glycoprotein was observed to disappear from the hemolymph of C. maenas during extended fasting and hemolymph seemed to be the site of storage of protein (Busselen, 1970). The concentration of hemolymph protein 66 decreased in the starved male lobster, Homarns americanus (Stewart, et al. 1967). In

Crangon vulgaris, starvation affected hemolymph protein contents and the protein markedly dropped after 23 d of starvation (Djangmah, 1970). Glycogen in midgut gland of the isopod, Lirceus brachyurns (Harger) was also used as an energy source during starvation (Steeves, 1963). In Ba/anus balanoides there was evidence that the first reserve used by starved animals was carbohydrate, followed by utilization of lipid and then protein, if the amount of carbohydrate dropped to 10 % of body weight

(Barnes, et al. 1963). In the snail Thais lamellosa, however, protein and carbohydrate were utilized during starvation after completion of spawning (Stickle, 1971 ).

Activities of both NAGase and chitinase in the present study were not significantly different between feeding and fasting crabs. It was presumed that the three days of fasting used was insufficient to alter the midgut gland conditions which retained near normal enzyme activity.

4.4.4. Kinetic properties

The apparent ~ value for the NAGase of Scylla employing p-NAG as substrate, was 0.11 ± 0.06 mmol.L1• Michaelis constants for the exochitinases of Manduca sexta, 0.25 ± 0.04 mmol.L- 1, in the cockroach, Periplaneta americana, 0.31-0.37 mmol.L1, and in the spider Cupiennius salei (Table 12) were similar to this value.

However, these values for this three species were considerably lower than those reported in the haemolymph (3.6 mmol.L1) and moulting fluid (6.3 mmol.L-1) of the silkworm, Bombyx mori, the krill (2.7 mmol.L-1) Meganyctiphanes norvegica, and

Drosophila hydei, 5.7 mmol.L1 (Table 12).

The Km value for chitinases in Scylla ( 4.32 ± 2.65) was in the same range as found in the krill, Meganyctiphanes norvegica (1.6 and 5.2 mg chitin.mL1), and the dipterans. Drosophila hydei 5 mg.mL1, D. melanogaster 2.2 mg.mL1 (Table 6). Kinetic 67 properties of chitinolytic enzymes from Scylla were similar to other arthropods. It was presumed that under these high (K,,, values) conditions the activity of enzymes was proportional to substrate and thus after feeding, when substrate concentration rose there would be automatic increases in enzyme activity (Krebs, 1972). In the present study the crude enzymes were used and purification of the enzyme would be expected to provide more precise data for kinetic values and specific activities.

4.4.5. Effect of temperature

The larvae of Scylla (zoeal stage) are inactive below 10°C (Hill, 1974). Adult crabs show decreased activity below 16°C and cease eating at 12°C (Hill, 1980).

Suitable water temperatures for larvae are between 20-24°C (Queensland of

Department Primary Industry, 1984) and they cease growing when temperature falls below 20°C. Juveniles are more tolerant of low temperature than the larvae and their ideal temperature is 18-30°C. They grow faster at higher temperatures within this range (Luo and Wei, 1986). Catchability of this species appeared to be influenced by temperature fluctuations as well. Catch per unit effort (CPUE) an assessment method in fisheries, decreased toward winter time and increased again in summer in two South African estuaries (Hill, 1975). A similar trend in seasonal catches was also found in Australian waters (Burke, et al. 1984). CPUE was low in the winter months

(June-July) and high in summer.

In the view of this, the temperature range selected for enzyme assays was 15, 20,

25, and 30°C for NAGase and at 15, 25 and 30°C for chitinase, as this covers the temperature range in which the crabs normally live. In the present study, the activity of both NAGasc and chitinase increased with increasing temperature up to the maximum used (30°C).

00 00 0--

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Spindler Spindler

Reference Reference

This This

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2.65 2.65

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(mg.L·

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Chitinase Chitinase

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6.3 6.3

0.02 0.02 0.04 0.04

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0.37 0.37

1.3 1.3

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5.7 5.7

0.35 0.35

0.25 0.25 3.6 3.6

P-N-acetyl-

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0.31 0.31

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cuticle cuticle

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juice, juice,

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constants constants

animals animals

blood, blood,

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moulting moulting

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Kc Kc

Whole Whole

Gut, Gut,

Foregut Foregut

Michaelis Michaelis

of of

norvegica norvegica

comparison comparison

americana americana

hydei hydei

salei salei

melanogaster melanogaster

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sexta sexta

mori mori

12. 12.

serrata serrata

Table Table

Cupiennius Cupiennius

Manduca Manduca

Bombyx Bombyx

Drosophila Drosophila

Drosophila Drosophila Periplaneta Periplaneta

Meganyctiphanes Meganyctiphanes

Scylla Scylla

Species Species

I I

8 8

7 7

5 5

6 6

3 3

2 2

No No 4 4 69

In other crustaceans studied, the chitinolytic enzymes also seemed to be dependent on temperature. The NAGase from Meganyctiphanes n01vegica had an apparent temperature optimum at 40°C, whereas in Euphausia superba it was 50°C

(Spindler and Buchholz, 1988). Both are from cold Antartic waters. NAGase from the Mediterrean krill, M. norvegica med, lived at an ambient temperature range 12-

130C, had temperature optimum at 30°C and 46°C. In the Kattegat krill, M. norvegica, (ambient temperature range 0-l5°C) types of NAGase were also present with temperature optima of 40°C and 53°C. The activity of NAGase from Ocypode ryderi, living at a temperature range of 25-40°C, had an apparent temperature optimal of 50°C. Two types of NAGase from Serolis polita, lived at temperature range -1.9°C to 2°C, had temperature optimum at 43°C and 50°C (Buchholz and

Vetter, 1993). The chitinase showed optimum activity at temperature of 40 and 45°C for M. norvegica, and E. superba, respectively. Both enzymes were inactived at 60°C

(Spindler and Buchholz, 1988). The optimum temperature for the enzyme activity in these species was very much higher than that the temperature at which live in and this is also evident in other enzymes of other cold-blooded animals. In the lobster

Nephrops norvegicus, for example, the apparent temperature optimum for amylolytic enzyme activity was about 57°C and the temperature of destruction at about 76-78°C

(Yonge, 1924 ). In Orc.:onectes virilis, the temperature optimum for proteolytic enzyme activity from the foregut juice was about 49°C (De Villez, 1965). In Scylla it is probable that maximal activity of the chitinolytic enzymes occurred at a temperature

> 30°C but it is unlikely that the species often experiences temperatures significantly higher than the 30° used in these experiments.

In general, metabolic rate will increase with increasing temperature and the rate

of incrcaSl' is commonly expressed at the Q10, the factorial change in activity for a l0°C change in temperature (Withers, 1992; Prosser, 1973 and Schmit-Nielsen, 70

1990). In this study, the 0 10 for chitobiase ranged between 2.28-3.62 for temperatures from 15-30"C whereas for chitinase the equivalent range was 1.82-2.65. These values were within the range common to many biological rate processes (1 to 4). A change by a factor of 2 or above are indicates involvement of chemical reactions, whilst

values closer to 1 indicate physical processes at work (Wilson, 1979). Changes in 0 10 values for enzyme reactions may be due to an alteration in amounts of enzymes or their cofactors or a change in their properties (Gordon, et al., 1968). In Scylla, both

chitinase and NAGase were sensitive to a change in temperature but the 0 10 for

NAGase was greater than that for chitinase. This ensured that NAGase activity was always in excess of chitinase activity so that there was no inhibition of chitinase activity due to the accumulation of reaction product. Chitinase is the rate limiting process in chitin digestion.

Data for small Scylla (10g) indicated a constant Q10 for oxygen consumption or 1.8-1.9 over the temperature range 16-38°C (Veerannan, 1972). If this holds for

the larger crabs used in this study then Q10 for metabolism will be similar to those for chitinase and NAGase at the lower end of the temperature range. However, as

temperature increases so does the Q10 for chitin digestion and the latter rise well above the value for metabolism. Thus at elevated temperatures Scylla could utilise greater amounts of chitin in the diet than at lower temperatures.

4.4.6. pH of digestion

In this study, NAGase exhibited 2 peaks of activity; a very large peak at pH 8 and a minor peak at pH 4 (Fig.7). The pH-optimum of NAGase varies among organisms studied. Using p-NAG as a substrate, the optimum was at pH 5 in

Meganyctiphanes non1egica and Euphausia superba (Spindler and Buccholz, 1988), pH

5.5 in Drosophila hydei (Spindler. 1976), pH 5.0-6.0 in Bomyx mori (Koga, et al. 71

1987).

It appeared that the optimal pH depended on a number factors, such as the assay condition, substrate, type of buffer used and species. In Periplaneta americana, for instance, the pH-optimum of NAGase was 4.6, when citrate buffers were employed and 5.1, if acetate buffers were used (Powning and Irzykiewiecz, 1964).

The pH-optimum of NAGase in the spider Cupiennius salei, using chromogenic substrates, was at pH 5.4, whilst optimal activity was found at pH 6.5, when using a natural substrate (Mommsen, 1980). In Bacillus subtilis, the crude enzyme was used to determine activity of NAGase and the pH-optimum was 5.9, whilst the purified enzyme exhibited an optimum pH of 5.9-6 (Berkeley, et al. 1973). In the stomach of marine fishes, activity of NAGase in the Dover sole, So/ea so/ea, using p-nitrophenyl

/3-N-acetylglucosaminide as a substrate, was very high at pH 5, but no activity was exhibited at pH 2-3 and pH 8-9 (Clark, et al. 1988). Activity of NAGase from the gastric tissue Squalus acanthias and Raja radiata possessed pH-optimum 4.5 and 4.25, respectively (Fange, et al. 1979).

Activity of the chitinase, using colloidal chitin as a substrate, showed a similar pattern to the activity of NAGase. Activity occurred over the pH range used from pH 4-9 with a peak at pH 8. This pH optimum falls within the range of values obtained from other arthropods, using colloidal chitin as a substrate. Activity of chitinase in the midgut gland of Penaeus japonicus, using chromogenic and colloidal chitin as a substrate, had pH-optima at 6. 7 (Kono, et al. 1990). In the moulting fluid of Manduca sexta, the optimum activity of chitinase was at pH 8.2 (Bade, 1974).

In fishes (Notothenia rossii marmorata, N. neglecta, Champsocephalus gunnari and

Chaenon!phalus aceratus) that feed on krill, Euphausia superba, the activity of chitinase. measured using a suspension of purified chitin, was very high in the stomach tissue (Rehbein, et al. 1986). In Chimaera monstrosa, a fish that feeds largely 72 on prawn, robust activity (using glycol chitin as a substrate) was displayed at pH 8-10 and a little activity at pH 3 in the pancreas. In the gastric mucosa of Coryphaenoides rupestris, the pH-optimum was 1.25, whilst Squalus acanthias possessed two peaks, a small peak at pH 3.6 and a longer peak at pH 1.6 (Fange, et al. 1979). High activity of chitinase was also found in the stomach and intestine of juvenile Dover sole, Solea solea, with an optimum pH at 2.2 and 5, respectively (Clark, et al. 1988).

The alkaline chitinolytic enzyme activity at pH 8-9 of Scylla was probably of bacterial origin?, since chitinolytic bacteria was found in a penaeid shrimp (Dempsey and Kitting, 1987) and fish (Goodrich and Morita, 1977). The results of this study showed that more than one peaks (pH 6 and pH 8) were observed which perhaps indication of more than one chitinase or NAGase were present in the foregut fluid and midgut gland. The presence of more than one chitinase and NAGase has also been found in other invertebrates. In the foregut juice of Homarus americanus, for instance, two NAGase and three chitinases are present (Lynn, 1990) whilst three chitinases were recorded from the whole body extracts of Meganyctiphanes no,vegica and Euphausia superba (Splinder and Buchholz, 1988) and three chitinases were also present in a cell line of Drosophila melanogaster (Boden, et al. 1985).

In the present study, the digestive juice was taken 2 h after meal and the enzyme activity was presumably representative of the second wave of secretory activity reported by Barker and Gibson (1978) in Scylla.

4.4. 7. Enzyme activity in the midgut gland

The midgut gland of crustaceans is also referred to as the hepatopancreas, hepatic gland. hepatic caecum, digestive gland, pancreatic gland, liver, branced midgut ceca. hepatopancreatic ceca, intestinal caecum, branccd diverticula. racemose gland, midgut diverticula. lateral caecum. midgut ceca, and pancreas (McLaughlin, 73

1983). The term 'midgut gland' is to be preferred and is used in this study (Van

Weel, 1974). Van Weel considered that the term 'hepatopancreas' was inappropriate since the organ had dual functions; ability to produce digestive enzymes and to absorb the digested food. Wether liver (hepato) nor pancreas can perform both this functions. The organ is comprised of large tubules and fills most of the cephalothorax cavity (Barker and Gibson, 1978). The tubules consist of an epithelial cell longer composed of four cell types; the E-( embryonic), F-(fibrillar), R-( absorptive), and B

(secretory) cells (Momin and Rangneker, 1974; Gibson and Barker, 1979). Digestive enzymes are produced by midgut gland F-cells and B-cells released to the lumina of

the tubules. There they may function in intraluminal digestion or the digestive juices

may be pumped into the posterior chamber of foregut, via dorsolateral grooves of posterior chamber, to mix with fluid extracted from the food by the muscle contraction mechanism. Some juice is driven to the anterior diverticulum of the

midgut and the mixed juices are then passed to the anterior chamber of the foregut

as well (Barker and Gibson, 1978; Dall and Moriarty, 1983). In Homarus gammarus,

digestive enzymes are fabricated by F-cells and liberated by secretory B-cells. The

activity of enzymes was maintained at 0-15 min, 1-2 hand 3.5-5 h, respectively, after

feeding (Barker and Gibson, 1977).

The application of protease inhibitors had no significant effect on activity of

NAGase or chitinase in this study (P>0.05). In the foregut juice of lobster, Homarus

americanus neither chitinase nor NAGase were inhibited by

phenylmethylsulfonylfluoride (Lynn, 1990). The lack of effect of protease inhibitor in

the enzyme activities from Sly/la indicated the absence of proteases with serine

residues in their active sites, like as reported in H. americanus (Laycock, et al. 1989;

Lynn. 1990). in the digestive gland of Panulirus japonicus ( Galgani and Nagayama.

1987). and in the digestive juice of Orc:onectes virilis (DeVillez, 1965). 74

Specific activity of NAGase measured in digestive gland extracts of Scylla in the present study (7.60 - 88 mmol.L1.min- 1.mg- 1) appeared to be higher than that of the

NAGasc activity measured for Drosophila hydei, 16-20 mmot.L·1.min-1.mg-1 protein

(Spindler, 1976). Specific activity of NAGase and chitinase in the integument of the krill, Euphausia superba changed markedly during the moulting cycle (Spindler,

1983 ). In the prawn, Palaemon se"atus, NAGase and chitinase activities in the midgut gland were high during moulting cycles and increased at premoult stage D1

(Spindler, et al. 1990).

In conclusion, this study showed that chitinolytic enzymes from the foregut fluid and midgut gland of Scylla were similar to those of other arthropods. Although the ecological significance is unknown, the regulation of chitin degradation seemed to be depend on the pH in gut and temperature where the crabs active. Chitinase behaved as an endo-type chitinolytic enzymes which split colloidal chitin very slowly. It appeared that the association of NAGase and chitinase was well adapted to function as a digestive enzyme. Further purification of foregut fluid and midgut gland extract arc needed to determine how many types of chitinolytic enzyme are present in this species. The importance of chitinolytic enzymes is a valuable indicator of the feeding habits of Slylla indicating appreciable intake of chitin in its normal diet. This study indicates that digestion and utilization of dietary chitin would occur and might be expected to make a significant nutritional contribution to Scylla. This is supported by data from the assimilation study (Chapter 2). 75 General Summary

1. Assimilation of dry matter, nutrients and energy.

The AAC of dry matter, protein and lipid chitin and exceeded 90%, 98%, 94% and 96% respectively. Values for utilisation of total energy in the diet and energy derived from protein and lipid were more than 99%, 97% and 93%, respectively.

Both bivalve and prawn diets were highly assimilated by Scylla and protein was the principal component to the diet. In the assimilation trials, faeces were collected in a plastic pouch. To check if bacterial decay of faeces affected assimilation data, antibiotics were included in the pouch on prawn diet 2 as a control treatment. There were no significant differences in assimilation of dry matter, nutrients and energy between control and experimental treatments which indicated that there was no bacterial degradation of the faeces during their residence in the plastic collection bag. The collection technique for faeces used in the present study gave satisfactory

results and allowed use of the direct method of measurement of assimilation in an aquatic environment.

2. The rate of passage of foodstuffs through the digestive tract.

57 -Co and PEG labelled microspheres fulfilled the requirements of particle and

fluid digesta markers in the digestive tract of Scylla. The particle marker was excreted

86.66% ± 7.99% of total dose at 308.17 h. The MRT of particle marker was 19.16 h

± 6.89 h. However, only 12.51 % of total injected PEG had been recovered 648 h

after injection. Use of fluid markers in the crustacean gut is inappropriate due to the

recirculation of fluid between midgut and foregut. 76

3. Chitinolytic enzymes from the foregut fluid and midgut gland

The pH of foregut fluid of Scylla ranged from 5.5 to 6.8 and fell within the range measured in other decapod crustaceans. Three days fasting did not alter enzyme activity which retained near normal enzyme activity. Kinetic properties of chitinolytic enzymes from the foregut of Scylla did not differ greatly from those other arthropods. The activity of chitinolytic enzymes from foregut fluid was dependent on the pH in the gut and NAGase and chitinase showed maximal activity at pH 8.

Enzyme activity also increased with increasing temperature in the range examined 15-

300C. Protease inhibitors had no significant effect on activity. The activity of chitinolytic enzyme from midgut gland was similar to the activity of enzymes from foregut fluid. 77 CONCLUSIONS

The nutrient composition of bivalve and prawns was highly assimilated by the crabs indicating that natural animal diets were utilized efficiently by adult Scylla.

Protein was the major source of energy available to crabs on the experimental diets.

The markers 57-Co labelled microspheres and PEG were not absorbed in the midgut gland cells and their behaviour in the digestive tract was suitable for their purpose. 51 -co was an appropriate particle marker for Scylla but fluid markers are not suitable for the 'recirculatory' digestive system of crabs.

Chitinolytic enzymes functioned well as digestive enzymes in the conditions of pH and temperature experienced in the gut. NAGase and chitinase activities from foregut fluid of Scylla were not affected by three days fasting. The activity of enzymes was influenced the pH and temperature. 78 ACKNOWLEDGEMENTS

I would sincerely like to thank my supervisor Prof. Peter Greenaway, whose support, guidance, advice and constructive criticism have been of great benefit throughout the course of this work.

I would also like to thank Prof. A.M. Beal, School of Biological Science, who acted as a supervisor during Prof. Greenaway's absence overseas, Prof. T.J. Dawson,

School of Biological Science, for permission to use the laboratory, Dr. P.I. Dixon,

Director of Centre for Marine Science, for permission to use the Marine Laboratory,

David Hair, D.G. Varley, B. Chan and A. McLean for assistance with buying crabs and laboratory work.

I am indebted to the Australian Government, International Development

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Tower , <-- Tank -> I ' - I ..l ' I ! i Extra Extra Filter Filter u

Experimental ; IExperimental n; I ' I Tank Tank ! I \"" '- \\// P ■

> i i Main ,----I' Pool I ! '---v A B Filter Tank µI I

A = glass tube for aeration B = plastic tube for water supply

Experimental Tank ---> water flow P = pump Figure 2. Assimilation of nutrients

103 - Dry matter 102 □ Protein I o Fat 101 '7 -, □ Chitin - I C _J - 100 I CD (.) 99 b a ab -CD u0 98 C 97 0 b 96 E en 95 en < 94 b -C: CD 93 (U -a. a. 92 < 91 90

Bivalve Prawn 1 Prawn 2 Diet

(Values as Means i .SD; n•8 for bivalve diet and prawn diet 1; n•9 for prawn diet 2; Different letters Indicate significant differences between mean coefficients for particular nutrients on different diets P<0.05) Figure 3. Assimilation of Energy

104 □ Total Energy 103 □ Protein Energy 102 □ Fat Energy - § Chitin energy C: 101 -CD - a a a u 100 r:: a b ab -CD 99 0 u

C: 98 0 97

E 96 95 94 - -C: CD ... 93 "'a. a. c( 92 91 90

Bivalve Prawn 1 Prawn 2

Diet (Values as means z. &.D; n•S for bivalve and prawn diet 1; n•9 for prawn diet 2; Different letters indicate a significant difference between mean values for a nutrient on different diets p<0.05) Figure 4. Excretion of Particle Marker in Sey lla serrata

110 l------· --- -

I -C ~ 0 100 -...CD u 90 )( CD as 80 -0 - 70 - ~Co-57 -~ C 0 60 • -CD... u )( 50 w... CD 40 ! ~... ~ as ~ 30 CD > 20 -as ::::, E - ::::, 10 (.)

0 100 200 300 400 500

Time (h)

Values as Means ± SD; n= 6 for 57-Co; n=5 for PEG Figure 5. The Percentage of Digesta Markers Excreted in Scylla serrata

100 -r------, I ~ ' 90 1 ,------/ -,:, 80 -~ CD -() CD ·-c: ... 70 CD ~... 60 "'E ---00---- PEG --+- 57-Co -,:, 50 -~ CD -...CD () >< 40 w... CD ~... 30 ~"'

20 ~ i ✓ 10 -i /// : ,,m-----ID 0 ,f / ; · T'T--,-l'T 'I

0 100 200 300 400 500 600 700

Time (h)

Means n= 6 57-Co; n= 5 PEG CH20HOH i --CY\~ NH ~H 20H H -----....____ I H~L-r ~ 0~1~!_/ "O \ ~\/ I ~ / --0 HO____,__..-- \ ----..., Hr CH20H \,H H I I OC co I I I I H3C CH3

Fig. 6. The structure of chitin (after Muzzarelli, 1985) Figure 7. Diagram to illustrate enzymatic degradation of chitin

Chitin (polysaccharide)

endo

Oligomeric residues (Dimer, trimer, tetramer, tetramer, pentamer, hexamer) exo exo ;do

two-dimers

monomer dimer+ tetramer (monomer+ dimer) or two-trimers dimer + trimer (monomer + trimer)

exo = exochitinase endo = endochitinase Fig 8. The effect of pH on p-Nitrophenol

3.0 -r------~ ------~------· --~~------I I ~- / ~· / / , -E C 2.0 ■ -@-pH 3.8 0 ~ ---pH 4.8 "It' - ---0------p H 5.8 CD () -+-pH 6.8 C -D--pH 7.8 .a• ---pH 8.8 -0 U) .a 1.0 < • ----~-/----~ ::,-----

-~ iv--- / -- 7 • 0.0 I I 1=7 0 10' 20 30 40 50 60 70 80 p-Nitrophenol Cone. (µmol.L-1)

The absorbance of PNP in different pH at given concentrations; nm= nanno meter Figure 9. Activity of~ -N-acetylglucosaminidase in an individual sample

Abs Assay: NAGase in foregut juice

0.88

0.84

0.80

0.76

0.72

0.68

0.64

0.60 00:00 Time (mm:ss) 05:00

NAGase was assayed using p-NAG as a substrate in a universal {Mcllvaine) buffer pH 7 at 25°G for 5 min; Abs = absorbance at 410 nm t>

•. ,. V mu. 13.9

I, .350 -s 0 K,. .350 ic.a' .129

Fig. 10. The direct linear plot method (Eisenthal-Comish-Bowden method). Sample plot of the effect of concentration of substrate (p-nitrophenyl-N-acetyl-P-D glucosaminide) on the activity of P-N-acctylgJucosaminidase.

Determination or K,. and V_

The vertical axis (y) is used to determine the V_ value and the horizontal axis (x) is used for K.- The values for v are plotted against values for -s. The two points are then joined with a line which is extrapolated to the right of the graph. Where two lines intersect at the right, Km and Vmax arc represented by the coordinates of the intersection point. Mean Km (K.) and V ... (V...) arc calculated as the median of all intersections. Figure 11. NAGase Activity in Foregut Fluid Before and After Feeding

170 ------·------7

I I 160 --1 150 --7-- -G) I () I ·-:::, 140

G) 130 NS >

ti) 120 -G) CII) 110 -~ -.:::, 100 --I ...J 90 E 80 --I C: l ·- 70 E. 0 60 E =- 50 -~ ->, 40 -> 30 -() < 20 10 - 0 -----r------~T

Before After Feeding

Chitobiase was assayed using p-NAG as a substrate in a universal ( Mcilvaine) buff er pH 7 at 2!1' C; n=6; NS= not significant Figure 12. The activity of chitinase in foregut fluid before and after feeding

15 ------14 ----r-- -Q) - (.) 13 ·-::::, 12 ' NS Q) > 11 -Cl) Q) Q 10 "0 ..... 9 !..J E I~ ...... 8 I s:.. 7 Q -=- 6 >- 5 - -> 4 (.) <- 3 2 1 -l J J_ 0 ------T --- -

Before After

Feeding

Chitinase was assayed using colloidal chitin as a substrate in a universal (Mcllvaine) buffer lH 7; All sample were incubated for 20 h at 25 C; n•6; NS• non significance Figure 13. NAGase activity in foregut fluid fasting and feeding

r .. ---~------·------...... , 170 I 1 160 -,..--- 150 -CD () 140 j ·-::, 130 CD NS > 120 -j -U) i CD 110 c,, j ....."0 100 _.I 90 E ..... ~· I 80 C ·-E 70 0 60 -I §_ 50 ->, 40 -> -() 30 c( 20 10 0 i -1 I Feeding Fasting

Condition

Chitobiase was assayed using p-NAG as a substrate ig a universal (Mcllvaine) buffer pH 7 at 25 C; N• 6; NS• non significance Figure 14. The activity of chitinase in foregut fluid before and after feeding

15 r--·· -- j 14 j 13 -,I

- CD 12 -~I (.) NS ·-::, -i ·- 11 CD > 10 -u, CD ic:,) 9 "'C ..... 8 '...1 ...... E 7 6 - 5 -> 4

(.) <- 3 ~ 2 1

0 I Feeding Fasting

Condition

Chitinase was assayed using colloidal chitin as a substrate in a universal (Mcllvaine) buffer pH 7; All samples were incubated for 20 h at 25 °c n• 6; NS• non significance Figure 15. The effect of pH on NAGase activity

110 --,

100 ~ 90 I >. T - 80 I > 1 1 -(.) ! as 1 70 I : I as I E 60 ----< >< ~ as i I E 50 ---< I -0 40 c/!. 30

1 I ---, 20 I -1 I I I I -• -----, 10

0 ,- __ Lf L I -r 4 5 6 7 8 9 pH

Chitobiase was assayed using p-NAG as a substrate in a universal buffer ( Britton-Robinson)system at 25° C;n• 6. Figure 16. The effect of pH on chitinase activity

110 7

17 I 90

80 -1 ! -l -> 70 _j u -as E 60 :::, E >< as 50 E -0 40

4 5 6 7 8 9

Chitinase was assayed using colloidal chitin as a substrate in a universal (Britton-Robinson) buffer at 25° C;n• 6 Figure 17. The effect of temperature on NAGase activity

80 -> -cau 70 ca E 60 >< ca E 50 -0 40

I 0 T_J __ _ I I 3O0c Temperature

Chitobiase was assayed using p-NAG as a substrate in a universal (Mcilvaine) buffer pH 7 at given temperature; n• 6 Figure 18. The effect of temperature on chitinase activity

110 ------·-·------~-- 100 -- 90 >,. -> 80 u -j -as 70 E = 60 -1 E -, >< I as 50 E

-- ' ,-I_ 40 -0 '#. 30

20 -- ' I -1 10 I _L_,JI j __ 0 I I 1soc 2s0c 300C

Temperature Chitinase was assayed using colloidal chitin as a substrate in a universal (Mcllvaine) buffer at pH 7; All samples were incubated for 20 h at given temperature; n• 6