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Aquaculture 250 (

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

0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2005.03.012

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: djohnston@fish.wa.gov.au (D. Johnston).

Australian abalone aquaculture is an expanding

industry focused on the commercially important

blacklip (Haliotis rubra Leach) and greenlip (Haliotis

2005) 341–355

D. Johnston et al. / Aquaculture 250 (2005) 341–355342

laevigata Donovan) species (Shepherd and Hearn,

1983; Hahn, 1989b; Dunstan et al., 1996). Abalone

growers culture naturally occurring diatoms on settle-

ment 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. Vari-

able 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 varia-

tion 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 asso-

ciated 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 settle-

ment, 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 tran-

sitions 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 loopat 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 concentra-

tions of digestive enzymes are indicative of shifts in

the ability to hydrolyse dietary components and have

D. Johnston et al. / Aquaculture 250 (2005) 341–355 343

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 develop-

ment 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 encrust-

ing 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

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 (approx-

imately 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 identi-

fied 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 collec-

tion 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

D. Johnston et al. / Aquaculture 250 (2005) 341–355344

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 micro-

scope 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 Tri-

chrome 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 centri-

fuged 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 con-

ditions 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 Almicro-assays) were performed in duplicate at 37 8C(a-glucosidase, h-glucosidase and laminarinase) or 32

8C (lipase and trypsin) in IWAKI flatbottom micro-

plates 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-U-nitroanalide (BAPNA) dissolved in dimethylforma-

mide (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 U-nitroanalide 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-glucopyrano-side (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 U-nitrophenol was measured at A400. The molar

extinction coefficient is 18 300 M�1 cm�1 for U-nitrophenol at pH N9 (Erlanger et al., 1961).

Laminarinase activity was measured using lami-

narin 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

D. Johnston et al. / Aquaculture 250 (2005) 341–355 345

measured by adding this to glucose (HK) assay

reagent (Sigma G2020). Glucose (HK) reagent con-

verts 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 4-

nitrophenyl caproate (4-NPC) dissolved in ethanol

as substrate. Each assay contained a final concen-

tration of 2.5 mM 4-NPC in 6 mM sodium taurocho-

late, 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 extinc-

tion 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 one-

way 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. Sig-

nificant differences among means were determined

using Tukeys HSD post-hoc test (P b0.05).

3. Results

3.1. Ontogenetic changes

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

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.

3.1.2. 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

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).

D. Johnston et al. / Aquaculture 250 (2005) 341–355346

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

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.

D. Johnston et al. / Aquaculture 250 (2005) 341–355 347

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 through-

out all regions of the intestine. The columnar

epithelial cells of intestine IV and V were charac-

D. Johnston et al. / Aquaculture 250 (2005) 341–355348

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

Fig. 3. Transverse sections (TS) through the digestive gland of abalone st

demonstrating tubule structure and epithelial cell types. Scale, 100 Am. (B)

Scale, 25 Am. (C) TS showing cell types and duct cell budding in 102 day

budding in 137 days PS abalone. Scale, 25 Am. (E) TS showing tubule stru

TS showing lack of duct cell integrity and protein accumulation in 137 d

budding in the distal tips of duct cells; IIY, used to indicate stage II buddi

cell; Lu, lumen; Tu, digestive tubule.

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

ained with Mercuric Bromophenol Blue. (A) TS of digestive gland

TS showing cell types and duct cell budding in 80 days PS abalone.

s PS abalone. Scale, 25 Am. (D) TS showing cell types and duct cell

cture and density in 137 days PS bruntQ abalone. Scale, 100 Am. (F)

ays PS bruntQ abalone. Scale, 25 Am. IY, used to indicate stage I

ng; IIIY, used to indicate stage III budding; Cc, crypt cell; Dc, duct

D. Johnston et al. / Aquaculture 250 (2005) 341–355 349

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 accumu-

lation 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)

0

0.02

0.04

0.06

0.08 α-glucosidase

80 100 120 140 1600

40

80

120

160

200

C)

µmo

l.min

-1.m

g-1

Lipase

10

20

30

40

50

E)

Laminarinase

wild

artificial

Days post-settlement

wild

artificial

ab

c c

ab b b

xx

y

artificial

wild

ab b

cxx

y

xx

x

Fig. 4. Average specific enzyme activity for laminarinase (A), h-glucosidaa–c refer to the ANOVA comparing among the age groups all fed diatoms

juveniles with those fed diatoms or artificial diet. Means with the same le

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 (Flaminarinase=38.58, df

3,12, P b0.001; Fh-glucosidase=4.50, df 3,12, P=0.025;

Fa-glucosidase=27.64, df 3,12, P b0.001). 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-

0

0.1

0.2

0.3

0.4

0.5

0.6β-glucosidase

B)

0

4

8

12

16

20D)Trypsin

wild

80 100 120 140 160

Days post-settlement

artificial

wild

artificial

a ab ab bx x

y

xx

y

se (B), a-glucosidase (C), trypsin (D), and lipase (E). The subscripts

, while the subscripts x and y refer to the ANOVA comparing wild

tter are not significantly different from one another.

D. Johnston et al. / Aquaculture 250 (2005) 341–355350

glucosidase (Fig. 4C), however, there was a signifi-

cant increase in activity between 80 and 102 days PS

abalone, after which time activity was constant.

Trypsin specific activity did not change signifi-

cantly 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 b0.001),

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-gluco-sidase (F =61.06, df 2,8, P b0.001), a-glucosidase

(F=430.04, df 2,8, P b0.001), lipase (F =79.21, df

2,8, P b0.001), and trypsin (F =212.42, df 2,8,

P b0.001), 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 vacuo-

lation, loss of cell integrity, epithelial sloughing, and

lack of visible nuclei (Fig. 2F), as well as decom-

position 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

D. Johnston et al. / Aquaculture 250 (2005) 341–355 351

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 develop-

ment 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

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 carbohy-

drates (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 h-glucans 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 struc-

tural polysaccharide degrading enzyme (McCandless,

D. Johnston et al. / Aquaculture 250 (2005) 341–355352

1981) increased between 80 and 158 days PS also

reflecting the increasing rate of diatom consumption in

older abalone and subsequently macroalgae. In con-

trast, 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 (Serviere-

Zaragoza 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 identi-

fying 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 composi-

tion 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, carbohy-

drate, 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 diges-

tive 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

D. Johnston et al. / Aquaculture 250 (2005) 341–355 353

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 presum-

ably because both the natural (micro- and macroalgae)

and artificial diet of abalone contain mostly carbohy-

drate (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 detri-

mental 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 nutri-

tionally 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.

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 avail-

ability 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, Tasma-

nia, 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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