Development of the radula and digestive system of juvenile blacklip abalone (Haliotis rubra): Potential factors responsible for variable weaning success on artificial diets

Aquaculture 250 (2005) 341 – 355 www.elsevier.com/locate/aqua-online

Development of the radula and digestive system of juvenile blacklip abalone (Haliotis rubra): Potential factors responsible for variable weaning success on artificial diets Danielle JohnstonT, Natalie Moltschaniwskyj, Jarrod Wells School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia Received 7 December 2004; received in revised form 9 March 2005; accepted 9 March 2005

Abstract We investigated the structural and physiological changes in the radula and digestive system in juvenile blacklip abalone Haliotis rubra, between 80 and 158 days post settlement (PS) to determine if variable growth on artificial diets (and prevalence of runts) are due to an inability to efficiently ingest and digest the diet. Between 80 and 102 days PS, L5 teeth appeared on the radula and there were fewer lateral serrations, consistent with the adult form of the animal, suggesting that this development is in preparation for feeding on macroalgae. Digestive gland complexity (tubule number and density) increased between 80 and 102 days PS and is consistent with greater enzyme production and increased digestive efficiency. Of the enzymes studied, laminarinase and lipase exhibited the highest activities in animals fed a diatom diet, both significantly increasing with age of the abalone. High laminarinase activities reflect higher utilisation of the algal polysaccharide chrysolaminarin in the diatom diet. Ingestion of artificial diet had no adverse effects on the morphological development of the digestive system, but trypsin activity in abalone fed the artificial diet was significantly higher than diatom-fed abalone of similar age, indicative of higher levels of protein in the artificial diet. Similarly, lipase activity was significantly lower in abalone fed the artificial diet and may reflect an inability to digest the fish oil component, which is not found in their natural diet. Future development of artificial diets, especially for juvenile abalone, should focus on the levels and type of lipid provided. Runt abalone (i.e. under-developed compared to their siblings) had radulae similar to much younger 80 days PS abalone and digestive tissue degradation at 137 days PS is evidence that runts have limited ability to ingest food and are nutritionally compromised. D 2005 Elsevier B.V. All rights reserved. Keywords: Abalone; Weaning; Artificial diet; Digestive enzymes; Radula; Digestive system; Aquaculture

1. Introduction T Corresponding author. WA Marine Research Laboratories, P.O. Box 20, North Beach, Western Australia 6020, Australia. Tel.: +61 8 92468460; fax: +61 8 94473062. E-mail address: [email protected] (D. Johnston). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.03.012

Australian abalone aquaculture is an expanding industry focused on the commercially important blacklip (Haliotis rubra Leach) and greenlip (Haliotis

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laevigata Donovan) species (Shepherd and Hearn, 1983; Hahn, 1989b; Dunstan et al., 1996). Abalone growers culture naturally occurring diatoms on settlement plates as diets for newly settled juveniles, however, maintaining sufficient diatom numbers as the juveniles grow is a major obstacle in production (Ebert and Houk, 1984; Hahn, 1989a; Knauer et al., 1996). As juveniles increase in size and are ready to switch from diatoms to macroalgae farmers have two choices; provide natural macroalgae or use an artificial diet (Hahn, 1989a; Dunstan et al., 1996). Providing sufficient macroalgae is time consuming and expensive with supply from wild harvesting of local macroalgal beds unreliable and environmentally unsustainable (Hahn, 1989a; Dunstan et al., 1996). Consequently, growers wean abalone onto artificial diets that are cheaper and may produce faster growth if they meet the nutritional requirements of abalone better than an individual algal species (Britz, 1996a,b; Viana et al., 1996). When using artificial diets growers need to develop a successful weaning protocol, one element of which is identifying the age or size that abalone are able to consume and digest artificial diet successfully. Variable weaning success is a major problem for the industry and results in starvation and slow growth, with some individuals displaying only 20–30% of the average growth of their siblings (Hahn, 1989a). Differential growth results in considerable size variation within a cohort, which requires costly and labour intensive grading of the animals. The reasons for variable growth are unclear, and may in part be due to a poor understanding of changes in structure and function of the abalone digestive system during development and how these changes may be associated with changes in diet. The nocturnal and cryptic habit of juvenile abalone (Shepherd, 1973; Saito, 1981) make it difficult to observe changes in behaviour that are likely to be important in determining their patterns of mortality and growth (McShane, 1992). This has resulted in little being known about ontogenetic development associated with diet changes from microalgae to macroalgae. Immediately after settlement, abalone post-larvae feed on biofilm and mucus trails (Shepherd, 1973; Saito, 1981) and once the radula is developed, at around 0.8 mm shell length (SL) (Kawamura et al., 2001), juveniles start feeding

on diatoms, turf, and crustose coralline algae (Dunstan et al., 1996). Juveniles maintain this diet until they are large enough to undergo the final diet transition from diatoms to macroalgae (Jarayabhand and Paphavasit, 1996; Kawamura et al., 2001). The size of individuals at this final transition varies among species, ranging from 5 to 10 mm for Haliotis discus hannai (Kawamura et al., 2001), 7 to 8 mm for Haliotis rufescens (Hahn, 1989a) and 10 to 20 mm for Haliotis asinina and Haliotis ovina (Jarayabhand and Paphavasit, 1996). It is thought that the diet change occurs because increasing diatom consumption of growing abalone results in longer foraging trips (Shepherd and Turner, 1985). This in turn increases energy expenditure associated with locomotion making it necessary to exploit higher energy macroalgae as a food source (McShane, 1992). However, developmental changes in structure and function of the digestive system may also possibly influence the timing of dietary transitions and therefore weaning success. Changes in radula structure and organisation in Haliotis iris and H. discus hannai are thought to be in preparation for the feeding transition from micro- to macroalgae (Roberts et al., 1999; Kawamura et al., 2001). There is currently no information on the development of the radula and gut of blacklip or greenlip abalone cultured in Australia. The basic structure of the adult abalone digestive system has been described by Harris (1994). It consists of an oesophagus that extends posteriorly of the buccal region to a large crop organ, where food is stored before entering the stomach. A crop extends posteriorly into the stomach, which forms a 1808 loop at the posterior end of the abalone to extend anteriorly adjacent to the crop. A voluminous digestive gland, overlying the crop and stomach, occupies most of the visceral mass and is connected with the other digestive organs by a network of tubules and ducts. The stomach continues anteriorly extending into the style sac with the protostyle. The style sac is connected to a long and complex intestine with five regions (I–V). Food from the style sac enters region I and exits region V (also known as the rectum) which terminates at the anus. Ontogenetic changes in the types and concentrations of digestive enzymes are indicative of shifts in the ability to hydrolyse dietary components and have

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been used to identify dietary shifts in a range of invertebrates (Hammer et al., 2000; Johnston, 2003). For example, changes in protease and a-amylase activities indicate changes in feeding preferences of the black tiger prawn Penaeus monodon from herbivory in nauplius, to carnivory in mysis, and omnivory in juveniles and adults (Fang and Lee, 1992). Similarly, in Macrobranchium rosenbergii enzyme activity changes and hepatopancreas development occurs when individuals shift from exclusive carnivory to omnivory during the larval period (Kamarudin et al., 1994). Although this approach is common in invertebrates there is no quantitative data on ontogenetic changes in digestive enzymes in abalone which may assist in identifying the critical ages and sizes in relation to dietary changes. In this study we describe the structure of the radula and digestive system and document the digestive physiology (digestive enzyme activities) of juvenile blacklip abalone H. rubra between 80 and 158 days post settlement fed on diatoms. We also compare these structures and digestive physiology with abalone of the same age weaned onto artificial diet and with runts. This information will (1) identify changes in structure and/or physiology associated with dietary shift from diatoms to macroalgae, (2) determine when the digestive physiology of juvenile blacklip abalone is best suited to the transition from a natural diatom to artificial diet and (3) identify whether the digestive physiology or structural development in these runts can explain reduced growth.

2. Materials and methods 2.1. Sampling Juvenile H. rubra were collected from stock produced and reared at ABTAS Seafoods, Garden Island, Tasmania. Settled juveniles were fed encrusting diatoms and biofilm for 158 days. To document developmental changes in the structure and function of the digestive system abalone from the same cohort were sampled at 80 days (meanFS.E. maximum shell length, 3.5F0.04 mm), 102 days (8.5F0.06 mm), 137 days (10.5F0.11 mm), and 158 days (11.3F0.13 mm) post settlement (PS). Juveniles were selected from individuals at the top end of

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the size range to ensure that growing juveniles were sampled. At 137 days PS small juveniles (5.5F0.37 mm) in the cohort identified as bruntsQ (approximately 50% smaller than larger individuals) were sampled on the assumption that these individuals were not growing normally. To determine the effects of weaning on the digestive system, juvenile abalone weaned onto an artificial diet were compared to juveniles of the same age maintained on diatoms. At 105 days PS juveniles from the same cohort were transferred to a clean tank with no diatom biofilm and given an artificial diet manufactured by Adam and Amos, Mt Barker, South Australia. Abalones were fed the diet for 57 days until 162 days PS and 58 juveniles were sampled for histology and enzyme analysis. Only successfully weaned individuals were sampled, these were identified by a coloured band of new shell growth associated with the artificial diet. To fully assess the success of the artificial diet we compared the animals fed the artificial diet with starved animals. To starve juveniles, 20 individuals at 142 days PS were moved into a clean tank and maintained for 20 days without food. These animals were kept in the dark to ensure no algal film grew to provide a food source for these juveniles. The structure of the digestive system of starved juveniles was examined using histological and histochemical techniques. All animals were sampled early in the morning to ensure enzyme activity was at its greatest after they had been feeding during the night. Following collection abalone were transported in seawater or on ice for dissection in the laboratory. Prior to dissection or freezing maximum shell length (SL) of all abalone was measured to the nearest 0.1 mm using callipers. Wild juvenile abalone (20–30 mm) were collected from southern Tasmania and transferred to liquid nitrogen for storage within 8 h of collection. Digestive enzyme profiles of wild abalone were compared to the cultured abalone. 2.2. Radula structure Whole abalones were fixed in 10% seawater formalin for 24 h and the radula removed with forceps. The radula was placed in 1.5% sodium hypochlorite (NaOCl) for 1 h to dissolve attached connective tissue and mucous, then washed with

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distilled water and stored separately. Radulae were dehydrated in an ethanol series to 100%, critical point dried, gold sputter coated, and examined on an Electro Scan 2020 environmental scanning electron microscope at 15 kV. Morphology and the presence or absence of different tooth types (rachidian, laterals 1 and 2, laterals 3–5, and marginals) was described. 2.3. Histology and histochemistry Abalone were fixed in FAACC (10% formalin, 5% glacial acetic acid and 1.3% calcium chloride) for 24 h, after which the abalone tissue was removed from the shell and returned to fresh FAACC for storage at room temperature until processing. Whole abalones were processed for wax histology using standard methods and transverse serial sections (5 Am) cut and mounted on poly-l-lysine coated microscope slides. Sections were stained with Mallory-Heidenhain Trichrome and examined using an Olympus BH-2 microscope. Individuals used for histochemical analysis were fixed in 10% seawater formalin for 24 h and processed and sectioned as above. Sections of digestive gland, stomach, crop and intestine were stained with Mercuric Bromophenol Blue for 15 min at room temperature (Chapman, 1975). This stain binds free protein within tissue to identify where digestive enzymes were being produced and secreted. 2.4. Enzyme analyses The digestive gland from each abalone was dissected out on ice and pooled into one of four replicate tubes for each age group, the numbers pooled per age group depending on size of the abalone (80 and 102 days PS, n = 25; 137 days PS, n = 16; 158 days PS, n = 13; artificial diet n = 12). The digestive glands from wild abalone (12 in total) were dissected out on ice and four were pooled into each of three replicate tubes. Tissue was frozen in liquid nitrogen and stored at 80 8C until extraction. Thawed abalone were homogenised in chilled 0.1 M Tris 0.02 M NaCl buffer pH 7.5 using an Ultra Turrax homogeniser fitted with a S8N-8G dispersing tool (IKA-Works Germany). The homogenate was centrifuged at 10,000  g for 10 min at 4 8C and aliquots of supernatant stored at 20 8C.

One enzyme unit is defined as the amount of enzyme that catalysed the release of 1 nmol of product/min and was calculated using the appropriate molar extinction coefficient (e) for the assay conditions or standard curve. Specific activity was defined as enzyme activity per mg of abalone protein (units mg protein 1) and total activity was defined as enzyme activity per abalone (units abalone 1). Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Spectrophotometric enzyme assays (200 Al micro-assays) were performed in duplicate at 37 8C (a-glucosidase, h-glucosidase and laminarinase) or 32 8C (lipase and trypsin) in IWAKI flatbottom microplates and absorbances read in a Tecan Spectro Rainbow Thermo microplate reader. Appropriate controls were included with each analysis. Trypsin was assayed using N-a-benzoylarginine-Unitroanalide (BAPNA) dissolved in dimethylformamide (DMF) as substrate. Each assay contained a final concentration of 1.25 mM BAPNA in 0.1 M citrate 0.2 M phosphate buffer pH 5.5. Assays were initiated by the addition of enzyme extract and the release of Unitroanalide measured at A400–410. Under these assay conditions the molar extinction coefficient was 9300 M 1 cm 1 for U-nitroanaline (Stone et al., 1991). A positive control of 3 mg ml 1 porcine pancreas trypsin in 1 mM HCl was used. a-Glucosidase and h-glucosidase activities were determined using U-nitrophenyl a-d-glucopyranoside (Sigma N1377) and U-nitrophenyl h-d-glucopyranoside (Sigma N7006) as substrates, respectively. Each assay contained a final concentration of 4 mM substrate in 0.1 M citrate 0.2 M phosphate buffer pH 5.5. Assays were initiated with the addition of enzyme extract. Aliquots of assay mixture were removed at time intervals and added to 1 M Na2CO3 (pH 11), to terminate the reaction. Liberation of Unitrophenol was measured at A400. The molar extinction coefficient is 18 300 M 1 cm 1 for Unitrophenol at pH N 9 (Erlanger et al., 1961). Laminarinase activity was measured using laminarin as substrate. Each assay mixture contained 10 mg ml 1 of laminarin dissolved in 0.1 M phosphate buffer pH 5.5 and the reaction was initiated by addition of enzyme extract. Assay mixture was removed at 0 min and after 60 min incubation at 37 8C and the concentration of glucose liberated was

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outside of laterals, many marginals extended in pairs to the outermost edge of the radula tooth row (Fig. 1A,B). The rachidian tooth was slightly cuspid (curled tip), with the cusp towards the posterior end of the radula and its edge flattened (Fig. 1B). The 1st laterals interlocked with the rachidian and were different in morphology having a larger cusp (same direction) and a rounded spade shaped tip (Fig. 1B). The 2nd laterals were narrower than the 1st but also had a posterior facing pronounced cusp (Fig. 1B). There was a distinct change in tooth morphology between the 2nd lateral and the 3rd lateral. The 3rd lateral was the largest tooth in the radula and the tip (facing posteriorly) was extended and pointed, almost canine-like (Fig. 1C). The 4th lateral shared the same general morphology as the 3rd, but smaller in size with serrations on both sides of the tooth, whereas the 3rd lateral only had serrations on the outer side away from the rachidian (Fig. 1C). The marginals were distinguishable from the 4th lateral by increased serrations on both sides of the tooth and a rounded end as opposed to the point of the 3rd and 4th laterals (Fig. 1C). Marginals also orientate differently to the 3rd and 4th laterals being closely folded on top of adjacent marginals with their cusps pointing more outer laterally as compared to posteriorly. As the marginals moved to the outer edge of the radula the number of serrations on both sides increase. The radula of 102 days PS abalone had an additional 5th lateral that was the same shape as the 3rd and 4th laterals, but smaller in size (Fig. 1D). The rachidian was less cuspid (having a reduced curl in the tip) and the 1st and 2nd laterals appeared to increase in width. In addition to these changes the rachidian and 1st lateral of 137 days PS abalone had lost the cuspid shape and the 3rd, 4th and 5th laterals were larger and had sharper points. There was no change in the radula structure between 137 days and 158 days post settlement.

3.1.1. Radula structure At 80 days post settlement abalone had well differentiated teeth, typical of a Rhipidoglossan radula (Fig. 1A–C). The radula was asymmetrical with four types of teeth present in pairs within a tooth row with one either side of a central rachidian tooth; one rachidian, a pair of 1st laterals, a pair of 2nd laterals, a pair of 3rd laterals, a pair of 4th laterals. On the

3.1.2. Structure of digestive system In 80 days PS abalone the oesophagus was a longitudinally folded tubular organ (Fig. 2A), with an epithelium of two cell types; tall columnar ciliated, and mucus cells. Ciliation was sparse but extended around the entire internal perimeter of the organ (Fig. 2A). Longitudinal folding of the oesophagus increased between 80 days PS and 102 days PS and

measured by adding this to glucose (HK) assay reagent (Sigma G2020). Glucose (HK) reagent converts glucose in the assay mixture to NADH via a coupled enzyme reaction catalysed by hexokinase and glucose-6-dehydrogenase. After 15 min incubation at room temperature the absorbance of NADH, which is proportional to the concentration of glucose, was read at A340. The amount of glucose liberated min 1 mg 1 was calculated using a standard curve that was generated by incubating known amounts of glucose with glucose (HK) reagent. Lipase activity was determined using a method modified from Gjellesvik et al. (1992) using 4nitrophenyl caproate (4-NPC) dissolved in ethanol as substrate. Each assay contained a final concentration of 2.5 mM 4-NPC in 6 mM sodium taurocholate, 500 mM Tris, 100 mM NaCl buffer pH 7.4. Assays were initiated by the addition of enzyme extract and the release of nitrophenol was measured at A405. Under these assay conditions the molar extinction coefficient was 19,800 M 1 cm 1 for nitrophenol (Gjellesvik et al., 1992). 2.5. Statistical analyses Specific enzyme activity was compared among the age groups using a one-way ANOVA. A second oneway ANOVA was used to compare the enzyme activity among juveniles fed diatoms (157 days PS), juveniles fed artificial diet (162 days PS) and wild juveniles (unknown age). Data were tested for heterogeneity of variance using residual plots. Significant differences among means were determined using Tukeys HSD post-hoc test ( P b 0.05).

3. Results

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Fig. 1. Scanning electron micrographs of abalone H. rubra radula showing the type of teeth present within a tooth row. (A) Whole tooth row of 80 days PS abalone. Scale, 41 Am. (B) The middle of tooth row of 80 days PS abalone. Scale, 10 Am. (C) Transition of lateral teeth to marginal teeth of 80 days PS abalone. Scale, 13 Am. (D) Transition of lateral teeth to marginal teeth from 102 days PS abalone. Scale, 14 Am. (E) Whole tooth row from runt abalone 137 days PS. Scale, 28 Am. (F) Outer laterals of tooth row from runt abalone 137 days PS. Scale, 14 Am. R, rachidian (central tooth); L1, 1st lateral; L2, 2nd lateral; L3, 3rd lateral; L4, 4th lateral; L5 5th lateral; M, marginal(s).

ciliation increased along the ridges created by the folds, but with no change in cell structure. There was further increased folding in 137 to 158 day PS abalone (Fig. 2B). The crop of 80 days PS abalone had little epithelial folding with shorter columnar cells but during development folding increased with more prominent microvilli. The stomach epithelium of 80 days PS

abalone consisted mainly of columnar secretory cells with a cuticular lining, called the gastric shield and did not change during development (Fig. 2C). The style sac contained a highly ciliated columnar epithelium with granular inclusions in the distal tips and cilia around the entire distal region of the cells (Fig. 2D). The style sac did not change with development. Intestine I, III, IV and V were present

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Fig. 2. Transverse sections (TS) through the digestive organs of abalone stained with Mallory-Heidenhain Trichrome. (A) TS through the oesophagus of abalone 80 days PS showing epithelial characteristics. Scale, 25 Am. (B) TS through the oesophagus of abalone 158 days PS, showing extensive folding. Scale, 100 Am. (C) TS through the stomach of 80 days PS abalone showing epithelial characteristics and gastric shield. Scale, 25 Am. (D) TS through the style sac of 80 days PS abalone. Scale, 25 Am. (E) TS through intestine V showing epithelial characteristics and extensive folding. Scale, 25 Am. (F) TS through the crop of runt abalone 137 days PS. Scale, 100 Am. Bm, basement membrane; Ci, cilia; Cr, crop; Gi, granular inclusions, Gs, gastric shield; Mc, mucus cell; Mv, microvillus brush boarder; Nu, nuclei; Os, oesophagus; Sc, secretory cells.

in all stages of development, while intestine II first appeared in 158 days PS abalone contributing to an increase in length and complexity with abalone age. The intestine had a ciliated columnar epithelium although cell height and extent of ciliation differed between regions. Intestine II had a highly folded

ciliated columnar epithelium, whereas in intestine III cilia were short and sparse. Cells increased in height as they neared the typhlosole, a large food groove situated ventrally that extends longitudinally throughout all regions of the intestine. The columnar epithelial cells of intestine IV and V were charac-

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terised by the increased presence of mucus cells (Fig. 2E). Intestine V was highly folded and possessed the longest cilia in the intestine (Fig. 2E). The digestive gland of 80 days PS abalone was made up of a series of interconnecting tubules (Fig. 3A). Each tubule consisted of two epithelial cell types, duct and crypt cells, surrounding a central lumen (Fig. 3A). Duct cells were the most common cell type

within a tubule with crypt cells only occurring in small clusters (Fig. 3A). The entire cytoplasm of crypt cells stained blue (positive for protein) with Mercuric Bromophenol Blue, whereas only the distal cytoplasm of duct cells stained blue (Fig. 3A,B). Duct cells exhibited three stages of apocrine secretion into the tubule lumen (Fig. 3B,C). Stage I was characterised by small amounts of stain (protein) accumulation in

Fig. 3. Transverse sections (TS) through the digestive gland of abalone stained with Mercuric Bromophenol Blue. (A) TS of digestive gland demonstrating tubule structure and epithelial cell types. Scale, 100 Am. (B) TS showing cell types and duct cell budding in 80 days PS abalone. Scale, 25 Am. (C) TS showing cell types and duct cell budding in 102 days PS abalone. Scale, 25 Am. (D) TS showing cell types and duct cell budding in 137 days PS abalone. Scale, 25 Am. (E) TS showing tubule structure and density in 137 days PS bruntQ abalone. Scale, 100 Am. (F) TS showing lack of duct cell integrity and protein accumulation in 137 days PS bruntQ abalone. Scale, 25 Am. IY, used to indicate stage I budding in the distal tips of duct cells; IIY, used to indicate stage II budding; IIIY, used to indicate stage III budding; Cc, crypt cell; Dc, duct cell; Lu, lumen; Tu, digestive tubule.

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the distal tip; stage II by increased accumulation starting to form a distended bud; and stage III by a distinct bud at the distal tip or separation of the bud from the duct cell (Fig. 3B,C). The number of digestive tubules increased in 102 days PS abalone and duct cells showed more concentrated blue staining in the distal tips and more cells were budding into the lumen (Fig. 3C). There was no change in staining, location or prevalence of crypt cells within a tubule. There did not appear to be any change in tubule density within the digestive gland between 102 days PS and 137 days PS, although duct cells showed increased stain accumulation and budding in the distal tips as indicated by the increase in stage III duct cells (Fig. 3D). There was no change in digestive gland structure between 137 days PS and 158 days PS.

A)

50

3.1.3. Digestive enzyme activities All abalone 80–158 days PS fed on diatoms exhibited carbohydrase activity, however, activity levels varied among enzymes and age groups (Fig. 4). Laminarinase specific activity was the greatest of the tested carbohydrases for each individual age group followed by h-glucosidase and a-glucosidase (Fig. 4). All assessed carbohydrases increased in activity between 80 and 158 days PS ( F laminarinase = 38.58, df 3,12, P b 0.001; F h-glucosidase = 4.50, df 3,12, P = 0.025; F a-glucosidase=27.64, df 3,12, P b 0.001). Laminarinase specific activity increased by 86% between 80 and 137 days PS, after which there was no change (Fig. 4A). Specific activity of h-glucosidase gradually increased with age of abalone, with activity doubling between 80 and 158 days PS (Fig. 4B). The smallest activity of the five digestive enzymes assayed was a-

B)

Laminarinase

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D)

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Trypsin

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120 80 40 0

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Days post-settlement Fig. 4. Average specific enzyme activity for laminarinase (A), h-glucosidase (B), a-glucosidase (C), trypsin (D), and lipase (E). The subscripts a–c refer to the ANOVA comparing among the age groups all fed diatoms, while the subscripts x and y refer to the ANOVA comparing wild juveniles with those fed diatoms or artificial diet. Means with the same letter are not significantly different from one another.

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glucosidase (Fig. 4C), however, there was a significant increase in activity between 80 and 102 days PS abalone, after which time activity was constant. Trypsin specific activity did not change significantly from 80 days PS to 158 days PS abalone ( F = 2.52, df 3,12, P = 0.11). The average specific activity of trypsin for the four age groups was 1.02F0.017 units mg protein 1 (Fig. 4D). Lipase specific activity increased significantly from 80 days PS to 157 days PS ( F = 24.86, df 3,12, P b 0.001), with activity more than doubling (Fig. 4E). 3.2. Changes associated with weaning onto an artificial diet 3.2.1. Structure of digestive system In starved animals the number of tubules remained similar but the lumen area was enlarged and empty. Duct cells were smaller and showed minimal distal cytoplasm staining with no evidence of budding. Duct cells were often indistinguishable due to cell wall rupture and cell nuclei were not visible. In contrast, abalone that had been weaned onto artificial diet showed no visible differences in digestive system structure to those abalone fed diatoms of the same age. 3.2.2. Digestive enzyme activities In a comparison between juvenile abalone fed diatoms, artificial diet, and wild juveniles there was a significant difference in specific activity of h-glucosidase ( F = 61.06, df 2,8, P b 0.001), a-glucosidase ( F=430.04, df 2,8, P b 0.001), lipase ( F = 79.21, df 2,8, P b 0.001), and trypsin ( F = 212.42, df 2,8, P b 0.001), but not in laminarinase ( F = 3.45, df 2,8, P = 0.083). Abalone fed the artificial diet did not have significantly different specific enzyme activities in the five enzymes when compared to siblings fed on diatoms (Fig. 4). Wild juveniles had specific activities 3.6–8.7 times greater than those juveniles fed the artificial diet in all the digestive enzymes except for laminarinase (Fig. 4). 3.3. Runts 3.3.1. Radula structure The radula in runt abalone 137 days PS had only four lateral teeth (1st, 2nd, 3rd 4th) plus marginal

teeth, compared with the full complement of laterals in their larger siblings of the same age (Fig. 1E,F). The 137 days PS runts had a radula structure similar to that of full size 80 day PS abalone, with a narrow cuspid rachidian, 1st and 2nd laterals, and highly serrated 4th laterals (Fig. 1F). 3.3.2. Structure of digestive system There were fewer digestive gland tubules in runt abalone than in siblings of the same age, but crypt cells were similar in structure (Fig. 3E). Duct cells had lost structural integrity, with individual duct cells indistinguishable due to cell wall rupture and cell nuclei were no longer visible (Fig. 3F). There was also loss of epithelium from the basal lamina and very little accumulation of secretory products (blue staining protein) in the distal tips of duct cells (Fig. 3F). The epithelium of digestive organs in runt abalone was also very different to that of their 137 days PS siblings. Runts showed variable levels of degradation and tissue histology indicated severe epithelial vacuolation, loss of cell integrity, epithelial sloughing, and lack of visible nuclei (Fig. 2F), as well as decomposition of the foot muscle. Where the epithelium was identifiable, it had the same cell structure as 80 days PS abalone. Intestine IV was contorted into an irregular morphology.

4. Discussion 4.1. Ontogenetic development The radula structure of H. rubra juveniles was of typical Rhipidoglossan form: consisting of a central column of heavier central (rachidian) and lateral teeth, a fan-like arrangement of marginals on the outer perimeter, and made from superficially hardened chitin (Sollas, 1907; Crofts, 1929; Hickman, 1980). Major structural changes occurred in the radula of juvenile blacklip abalone H. rubra between 80 and 102 days PS. The rachidian of H. rubra was initially curled, but as the abalone aged the rachidian straightened resulting in a greater cutting ability by changing the angle that the tip makes contact with the substrate (Padilla, 1985). The development of L5 teeth and an increase in the size of L3–L5 suggests these teeth are more suited for gouging and collecting large

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food particles (Roberts et al., 1999; Kawamura et al., 2001). Fewer serrations on the outer laterals L3–L5 suggest that the radula has become less specialised for handling small food particles as the animal developed (Roberts et al., 1999; Kawamura et al., 2001). Increased grazing efficiency and capability of the radula with development in H. rubra is presumably needed for the transition to feeding on macroalgae allowing them to exploit larger and tougher food material and increase mastication efficiency. The radula of the older 102 days PS animals was similar to the adult form (Crofts, 1929; Herbert, 1990; Chitramvong et al., 1998; Roberts et al., 1999; Kawamura et al., 2001) and there were only minor morphological changes to the teeth after 102 days PS, suggesting that H. rubra are already fully equipped to consume a macroalgal diet by this age. The development of the radula in H. rubra was slower than other temperate species, such as H. iris and H. discus hannai, which have a full adult complement of 5 lateral teeth and associated tooth morphology by 60– 63 days PS (Leighton, 1974; Shepherd and Hearn, 1983; Roberts et al., 1999; Kawamura et al., 2001). The histology of individual digestive organs in H. rubra was similar to the adults of other abalone species, H. tuberculata (Crofts, 1929), Haliotis cracherodii (Campbell, 1965), H. rufescens (McLean, 1970; Bevelander, 1988) and H. laevigata (Harris, 1994; Harris et al., 1998). Elaboration of oesophageal folding between 80 and 158 days PS increases the epithelial surface area creating greater interaction between food particles and the cilia and the mucus, thereby increasing the rate of food passage into the crop (Roberts et al., 1999; Kawamura et al., 2001). Increased folding and expansion of the crop between 80 and 158 days PS would maximize the uptake of nutrients with more of the distal microvilli coming into contact with digesting food (McLean, 1970). The appearance of intestine II at 158 days PS greatly increases the area for nutrient absorption in older juveniles (Crofts, 1929; Campbell, 1965; McLean, 1970; Bevelander, 1988). This looped morphology slows the passage of food, increasing digestion and absorption (Oozeki and Bailey, 1995). Positive stain for protein in distal cytoplasmic granules in duct cells of the digestive gland indicates that these cells are producing and accumulating digestive enzymes. In contrast, crypt cells did not

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appear have an enzyme secretory function, given no distal accumulation of protein in the cells, no evidence of budding, and no difference in staining between fed and starved abalone. This is consistent with other studies that suggest duct cells synthesise enzymes capable of digesting proteins, lipids, and carbohydrates (Bevelander, 1988) and secrete enzymes to other organs such as the caecum (Purchon, 1968; Morton, 1979), crop (McLean, 1970) and stomach (Purchon, 1968; McLean, 1970; Morton, 1979). Hence the increase in the number of digestive gland tubules and digestive gland complexity as well as distal cytoplasm staining in duct cells in 80–102 days PS abalone indicates that digestive capacity increases with age. The enzyme secretory role of duct cells is evident in H. rubra, as enzymes are accumulated within the distal region of duct cells forming a bud before being released into the lumen. This is consistent with apocrine secretion described in other invertebrate species, e.g. the surf barnacle Tetraclita squamosa (Johnston et al., 1993). Juvenile H. rubra fed a diatom diet exhibited extremely high laminarinase activity reflecting the high concentration of the algal storage polysaccharide, chrysolaminarin, in the ingested diatoms. Wild abalone of similar age also had high levels of laminarinase suggesting that carbohydrases such as laminarinase play a major role in digestion for all abalone; a consequence of the high carbohydrate composition of their natural diet (Mori, 1953; Takami et al., 1998). Laminarin and chrysolaminarin are hglucans that are also present in brown macroalgae (Phaeophyta), and the same enzymes are necessary for the utilisation of macroalgae as a diet. Laminarinase activity increased from 80 to 158 days PS coinciding with changes in the radula structure, digestive gland, and digestive organs; supporting the hypothesis that these individuals are preparing for the shift in diet from diatoms to macroalgae. A similar pattern of development occurs in H. discus hannai, where laminarinase specific activity increased in conjunction with development of L5 teeth and differentiation of laterals in 63 days PS abalone (Takami et al., 1998; Kawamura et al., 2001). This would allow the dietary transition from microalgae to the macroalgae in this species (Takami et al., 1998; Kawamura et al., 2001). The specific activity of h-1,4-glucosidase, a structural polysaccharide degrading enzyme (McCandless,

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1981) increased between 80 and 158 days PS also reflecting the increasing rate of diatom consumption in older abalone and subsequently macroalgae. In contrast, a-1,4-glucosidase activity, more typically seen in terrestrial herbivores (Bennett et al., 1971), was low, approximately a 10th of h-1,4-glucosidase activity, and similar to the levels of a-1,4-glucosidase reported in adult H. rufescens (Bennett et al., 1971). There has been extensive research on the digestive proteases of Haliotis, compared to other enzymes, as industry pushes to incorporate higher protein levels into the diet (Fleming et al., 1996; Edwards and Condon, 2001). Trypsin activity in H. rubra did not change from 80 to 158 days PS, which does not exclude dietary transitions, rather reflects the low protein levels in both micro- and macroalgae (Viana et al., 1996; Edwards and Condon, 2001). Abalone cannot synthesise 10 of the 20 l-amino acids required to assemble proteins, and their growth is dependent on utilising what protein is available in the diet (ServiereZaragoza et al., 1997). Trypsin activities increase in adult H. fulgens, whereas juvenile H. fulgens lack carboxypeptidases A and B (Serviere-Zaragoza et al., 1997) and the carboxypeptidase-like enzyme of the adults (Hernandez-Santoyo et al., 1998). Future research on proteases of juvenile abalone approaching the age when diet transition takes place should focus on a suite of digestive proteases rather than an individual enzyme. Lipase activity in juvenile H. rubra is high compared with other enzymes and reflects the high content of lipid in their diatom diet, with a need to rapidly digest lipid rather than store it, as abalone contain only low lipid reserves (Dunstan et al., 1996). Lipase activity increased with age of H. rubra suggesting an increased ability to digest lipids as they develop and reflects the increase in lipid consumption with the higher diatom grazing rates of older abalone. Lipid digesting enzymes may prove useful in identifying a diet transition between wild juvenile and adult abalone, with a potential decline in activity indicating the utilisation of low lipid composition macroalgae (Ragan, 1981) as compared to higher lipid composition diatoms. In most cases, the digestive enzyme activities of wild H. rubra were significantly higher than in diatom or artificial diet fed cultured abalone of similar size. It is not clear what factors are responsible

for these differences. As the level of enzyme activity is a function of the diet it is likely that the composition of the diet differed rather that differences in quantity consumed. It may be that the natural diet ingested was considerably higher in protein, carbohydrate, and lipid than diatoms ingested by cultured animals. The exception to this trend was laminarinase activity which was not significantly different from cultured abalone. This suggests that the content of laminarin consumed in the diet of cultured and wild abalone was similar. It is clear that digestive enzyme analysis of wild abalone needs further investigation to fully understand the digestive physiology of these animals. 4.2. Artificial diet There were no differences in the morphology of the radula and the digestive system of H. rubra, feed natural and artificial diets; a similar situation for H. laevigata (Harris, 1994; Harris et al., 1998). There were however, significant differences in their digestive physiology. Artificial abalone diets are generally remarkably similar in their proximate composition, usually containing a high level of protein to increase growth rates (Viana et al., 1993, 1996; Britz, 1996a,b; Knauer et al., 1996; Bautista-Teruel and Millamena, 1999), a high level of carbohydrate and low levels of lipid (Fleming et al., 1996). Trypsin activity in the abalone fed the artificial diet was significantly greater than those of similar age fed diatoms, indicative of higher levels of protein in the artificial diet. Marine algae generally have a protein composition approximately 10–20% of dry weight (Piscos-Garcia et al., 2000; Edwards and Condon, 2001), whereas artificial diets have a protein content of 30–50% of dry weight (Fleming et al., 1996). This study showed juvenile H. rubra increased their trypsin activity to use the higher amounts of protein in the artificial diet. In contrast, adult H. rubra do not change trypsin activity in response to a high protein diet (Edwards and Condon, 2001) and it is possible that adult H. rubra do not share the flexible enzymology of the juveniles. However, adult trial was only for 3 weeks which may have been too short for a response. Juvenile H. midae also show increased protease activity after being fed an artificial diet (Knauer et al., 1996) and similar trends have been

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shown using more crude enzymatic methods for a number of other species on high protein diets, H. midae (Britz, 1996a); H. fulgens (Viana et al., 1996); H. discus hannai (Bautista-Teruel and Millamena, 1999). Feeding on artificial diet had no effect on the carbohydrase activities of juvenile H. rubra presumably because both the natural (micro- and macroalgae) and artificial diet of abalone contain mostly carbohydrate (Fleming et al., 1996). In contrast, lipase activity was significantly lower in abalone fed the artificial diet reflecting the decreased amount of lipid compared to diatoms. The natural diet of adult abalone contains only small quantities of fatty acids and lipids (Ragan, 1981; Dunstan et al., 1996). Artificial diets usually contain lipid at approximately 5% of dry matter (Fleming et al., 1996), as higher levels have detrimental impacts on growth (Dunstan et al., 1996). Unfortunately the investigation into lipid tolerance is limited to adults; interestingly juveniles may be better equipped to digest greater quantities of lipid. Certainly this study has revealed that juvenile H. rubra have a greater ability to digest lipid than previously thought and the level of lipid inclusion in artificial diets for blacklip abalone, H. rubra, may need to be revised to best reflect their lipid digestion capability. The type of lipid used also needs careful consideration, as the typical lipid sources for abalone artificial diets are fish oil and vegetable oil (Fleming et al., 1996), which are not utilised by abalone naturally. 4.3. Runt abalone Tissue degradation in under-developed (runt) abalone 137 days PS suggests that they were nutritionally compromised. Runts had radula similar to much younger (80 days PS) abalone and this lack of radula development and the resultant inability to ingest the adult diet may limit their survivorship. The under-developed radula structure of runt abalone may have been inefficient at removing diatoms, which had little or no adhesion to the substrate. The presence of food in the stomach is one of the main stimuli for enzyme secretion (Takami et al., 1998). Therefore, given that the duct cells had little or no enzyme budding present, and that minimal digestive enzyme activity was detected in these individuals it appears that runt abalone ingested little or no food.

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H. rubra runts will start growing again when removed from their siblings and fed an artificial diet (Hindrum, personal communication). Similarly H. fulgens exhibits compensatory growth when changed from a low nutrition value diet to one higher in nutritional value (Viana et al., 1996). Haliotis kamschatkana will also survive and make a full recovery after 27 days of starvation when feeding resumes (Carefoot et al., 1993). The actual cause of runting in these species is unclear, although availability or provision of the correct food appears to be a factor. Carefully controlled experiments that account for the effects of genetics, density, and feeding rates will provide clearer insights to causes of variability in growth rates. Grading individuals in each cohort and providing additional food and/or different food may be a way to manage stock to reduce variability in size caused by ineffective weaning of abalone juveniles from diatoms to artificial diet.

Acknowledgements We thank ABTAS Seafood, Garden Island, Tasmania, for supporting this project and supplying abalone for the duration of the experimental period. In particular, we thank Steve Hindrum and Andrew McArther for their co-operation and assistance. Thank you to Craig Mundy for collection of the wild abalone and Martin Lourey for critical review of a final draft of the manuscript.

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