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Effects of severe heat stress on immune

function, biochemistry and histopathology in
farmed Australian abalone (hybrid Haliotis
laevigata × Haliotis rubra)

Article in Aquaculture · August 2014

Impact Factor: 1.88 · DOI: 10.1016/j.aquaculture.2014.03.032

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 Aquaculture 432 (2014) 26–37

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Effects of severe heat stress on immune function, biochemistry and

histopathology in farmed Australian abalone (hybrid Haliotis
laevigata × Haliotis rubra)
Celia Hooper a,b,⁎, Rob Day a, Ron Slocombe c, Kirsten Benkendorff d, Judith Handlinger e, Julien Goulias f
a
Zoology Department, University of Melbourne, Parkville, Melbourne, Vic 3052, Australia
b
Gribbles Veterinary Pathology, 1868 Dandenong Rd, Clayton, Melbourne, Vic, Australia
c
Veterinary Pathology, Veterinary Clinical Centre, Princes Highway, Werribee, Melbourne, Vic 3030, Australia
d
School of Environmental Science and Management, Southern Cross University, PO Box 147, Lismore, NSW 2480, Australia
e
PO BOX 46, Kingsmeadows, Launceston, Tasmania 7249, Australia
f
IUEM, Technopôle Brest-Iroise, Rue Dumont d'Urville, 29280 Plouzané, France

a r t i c l e i n f o a b s t r a c t

Article history: High summer temperatures are one of the most stressful environmental problems confronted by the abalone
Received 22 September 2013 mariculture industry and are commonly associated with outbreaks of infectious disease. We tested the effect of
Received in revised form 12 March 2014 extreme but non-lethal elevated temperatures on abalone immunology, biochemistry and quantitative histology.
Accepted 21 March 2014
We subsequently compared the haemolymph results to the histology to gain increased understanding of how
Available online 12 April 2014
heat stress impacts abalone health. Abalone were kept in water that was heated from the ambient 16 °C temper-
Keywords:
ature to 26 °C within 5 h and then held at 26 °C for one week to determine the effects of this acute heat stress on
Abalone the day of temperature elevation and whether there was acclimatization or deterioration 2 and 7 days later.
Heat stress Antibacterial activity, phenoloxidase activity and neutral red retention times declined significantly with heat
Immunosuppression and did not recover. The total haemocyte count was elevated significantly during heat stress and was highest
Histopathology on day 1. The phagocytic rate was elevated on day 1 but had recovered by the following day. Acid phosphatase
Biochemistry activity, leucine aminopeptidase, haemolymph protein and haemolymph electrolytes (calcium, phosphate,
magnesium, sodium, and chloride) were not significantly affected by heat stress. This indicates that severe
heat stress causes changes in some, but not all haemolymph parameters. The sublethal immunologic effects
seen in haemolymph samples occurred concurrently with histological changes. The digestive gland had signifi-
cantly increased haemocyte infiltrates in heat stressed abalone. Heat stressed abalone had significantly greater
loss of epithelium lining from the gills, with no recovery. The gill goblet cell numbers declined significantly on
day 2 and had recovered by day 7. There was no significant change in the volume of fluid or protein concentration
of the haemolymph in the gill sinuses between treatment groups. These results indicate that immunosuppression
and organ damage are likely to be involved in the increased incidence of bacterial disease reported by abalone
farmers during summer.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Abalone aquaculture is an intensive industry, which is often land-
based, with tanks that are stocked at high density. Due to more crowded
conditions than in the wild (Goodsell et al., 2006; Shepherd, 1973) and
suboptimal environments, abalone in these systems are vulnerable to
infection by microorganisms that are often ubiquitous (Handlinger
et al., 2005). A link between stress and immunosuppression has been
established in abalone, associated with increased susceptibility to
⁎ Corresponding author at: Gribbles Veterinary Pathology, 1868 Dandenong Rd,
Clayton, Melbourne, Vic., Australia. Tel.: +61 3 95382271; fax: +61 3 95386741.
E-mail address: celia.hooper@gribbles.com.au (C. Hooper).
disease outbreaks (Cheng et al., 2004a; Hooper et al., 2007a; Wassnig
et al., 2009), including outbreaks of vibriosis associated with sharp
rises in water temperature during the summer months (Cheng et al.,
2004a; Handlinger et al., 2005; Reuter and McOrist, 1999; Travers
et al., 2008, 2009).
It would be very useful to the industry to be able to predict periods of
heightened stress on-farm that could lead to immunosuppression and
disease outbreak. Currently disease outbreaks are investigated after
they have occurred. Most diagnostic laboratory work in abalone is
based on histopathology and microbiology, either in routine surveillance
work to identify problems on a farm or in investigating the cause of
disease outbreaks (Handlinger et al., 2006; Hooper et al., 2007b;
Mouton, 2003; Pitcher et al., 2001). Prior to a disease outbreak, methods
are needed that will reveal stress sufficient to cause immunosuppression

http://dx.doi.org/10.1016/j.aquaculture.2014.03.032
0044-8486/© 2014 Elsevier B.V. All rights reserved.
 C. Hooper et al. / Aquaculture 432 (2014) 26–37
and thus predispose abalone to opportunistic bacterial infections. Two
approaches to this question are studied here with respect to the effects
of severe heat stress; haemolymph immune assays and quantitative
histology.
In vitro assays done on haemolymph that assess immune function in
abalone provide mechanisms for assessing their health status and
potential disease resilience (Dang et al., 2011; Hooper et al., 2007a;
Travers et al., 2008). Haemolymph sampling via the pedal sinus is a
non-lethal method to examine stock and some assays such as
haemocyte counts can be done rapidly on the farm (Hooper et al.,
2012). There has been a recent body of work on the effect of heat stress
on various immune and physiologic parameters in abalone. Haemocyte
counts were reported to rise quickly with sudden increased water
temperature and then decline as they acclimatized (Dang et al., 2012).
Lysosomal membrane stability (NRR assay) decreased with a sudden
decline or elevation from the temperature to which the stock had
been acclimatized (Wang et al., 2006). Phenoloxidase and phagocytic
rates declined after spawning during small temperature fluxes in
summer (Travers et al., 2008), but it was not clear what direct effect
water temperature had on these immune parameters, relative to
spawning stress. Antibacterial activity elevated transiently with heat
stress and then declined below baseline levels (Dang et al., 2012).
Leucine aminopeptidase (LAP), which is modified by bacterial disease
(Paillard et al., 2004), was not affected by temperature (Travers et al.,
2008). There have been no reports of the effect of temperature on acid
phosphatase (ACP) in abalone. Significant elevation of ACP in abalone
and other molluscs infected with bacteria has been found, indicating
that it is likely to be important in abalone immunity (Cong et al.,
2008; Wang et al., 2004).
The effect of thermal stress has not been tested on haemolymph
electrolytes in abalone. Furthermore, there have been only a few reports
on environmental stress and osmoregulation in abalone, including
decreased water pH (Burke et al., 2001), increased water nitrite concen-
trations (Harris et al., 1998), increased water salinity (Cheng et al.,
2002), and low dissolved oxygen (Cheng et al., 2004b) and these have
shown that different stressors have different effects on haemolymph
electrolytes.
Histopathology is an essential component of investigating disease
outbreaks and knowledge of histological changes seen with a common
stressor such as heat is needed. Knowledge of the lesions of heat stress
aids in differentiation of changes seen with infectious agents, feed
problems, toxic diseases and other causes, from those due solely to
heat stress. The reports that describe microscopic lesions associated
with environmental stressors include work on transport stress,
nutritional stress, overstocking (Mouton, 2003) and increased nitrate
in the water (Harris et al., 1998). There are a few reports on the effect
of heat stress on histology (Braid et al., 2005; Schaefer et al., 2013;
Travers et al., 2008). Heat stress alone leads to digestive gland atrophy
in a study extending over one year (Braid et al., 2005). Elevated heat
had varying effects on different levels of crop and stomach epithelium,
with none of the histological changes noted being associated with
altered growth rates (Schaefer et al., 2013).
There is a body of work comparing immune or metabolism assays in
haemolymph or tissues with histological changes in key organs in
abalone such as gill and digestive gland (Hooper et al., 2011, 2012;
Rosenblum et al., 2005, 2006; Zhou et al., 2010) but only Travers et al.
(2008) studied the effect of heat stress on histologic changes and
compared these with immune assays on haemolymph. Reports on
abalone histology that are quantitative (Braid et al., 2005; Friedman
et al., 2003; Harris et al., 1999; Moore et al., 2000; Rosenblum et al.,
2005, 2006; Vilchis et al., 2005) rather than descriptive, have indicated
that scoring allows statistical analysis of the results leading to increased
information gained.
Farm records show that the shallow on-farm tanks from which
the abalone were sourced, experience relatively rapid temperature
fluctuations, hence the rapid elevation of water temperature selected
27
for this experiment of 10 °C within 5 h. The only study of the critical
thermal maximum temperature (CTM) or upper lethal temperature in
Australian abalone found that 50% of Haliotis rubra and Haliotis laevigata
died at temperatures of 26.9° and 27.5 °C, respectively (Gilroy and
Edwards, 1998). The preferred temperatures for these two species
were 16.9 °C and 18.9 °C, respectively. Ectotherms acclimatized to
higher temperatures will tolerate higher temperature elevations than
those acclimatized to a lower temperature (Becker and Genoway,
1979; Cheng et al., 2004a; Portner, 2002). The acclimatization of
molluscs to elevated temperatures involves altering their metabolic
rate and can occur within 5–8 days (Peck et al., 2002). Water tempera-
tures of 26 °C have been recorded in land based abalone farms in some
parts of Australia and temperatures of 24°–25 °C are fairly commonly
recorded during summer. Farmers in these locations have reported
elevated mortality rates in previous years when the water flowing
through their tanks reached at least 24 °C for several consecutive
days. In this experiment, abalone were kept in water heated from the
ambient temperature of 16 °C to 26 °C for 7 days. The upper tempera-
ture and the rapidity of temperature elevation used were slightly
above reported temperatures and were selected to produce a severe
but non-lethal stress.
The effect of rising water temperature on farms is likely to be complex,
involving not only the effect of heat on metabolism but also environmen-
tal decline. Increased biofouling of pipes, decreased water quality in the
farm tanks, increased bacterial growth rates and up-regulation of the
expression of bacterial virulence factors such as adhesins are all affected
by rising temperatures (Cheng et al., 2004a; Parveen et al., 2008;
Rosenberg and Falkovitz, 2004; Toren et al., 1998). In this experiment
we imposed heat stress (and the consequent lower dissolved oxygen)
in a controlled laboratory environment that excluded these other
potential confounding factors occurring on-farm. This experiment
investigated whether abalone immunity, biochemistry and histology
were affected by sudden, severe temperature elevation and whether
there was acclimation or deterioration over seven days of continued
high water temperature.
2. Methods
2.1. Abalone
Juvenile abalone used in this study were two year old hybrids of
male H. laevigata × female H. rubra, sourced from a Victorian land-
based farm. Hybrid abalone are commonly farmed in Australia and the
work is thus relevant to commercial farmers of this species.
The abalone were acclimatized for two weeks at 16 °C, in 12 tanks
that contained 8 l of water and eight abalone. The water was
recirculated: after sand biofiltration, seawater was held in an 18,000 l
reservoir and passed through a 2 mm mesh filter. The flow rate through
each replicate tank was 10 l/h throughout the experiment.
Treatments were applied independently to each tank. Each tank was
saturated with oxygen by delivering air from a compressor via a plastic
airline to each tank, with the airline opening at the bottom of the tank.
The temperature increase will lead to a reduction in dissolved oxygen,
but this is what would happen on-farm in hotter conditions, so that
heat stress is regarded as involving the effects on abalone of these
combined changes. Feeding ceased the day before the experiment in
both control and treated tanks and all food and faecal debris were
removed. The tanks were covered to keep out light, except at the actual
time of sampling.
On day 1 of the experiment, heating rods were placed in 6 of the tanks
and over a period of 5 h, the water temperature was raised from 16 to
26 °C. This temperature was monitored twice daily by a thermometer.
Two abalone per tank were sampled on day 1, 5 h after the temperature
had been raised to 26 °C, followed by two more abalone on day 2 and
again on day 7. The abalone were observed as the temperature rose to
document any behavioural changes.
28 C. Hooper et al. / Aquaculture 432 (2014) 26–37
2.2. Haemolymph sampling, immune assays and biochemistry
Sampling involved manual removal of an abalone from each tank,
removing any water drips, then weighing it to within 0.1 g and taking
haemolymph from the pedal sinus within 1 min after removal from
the tank.
2.2.1. Total haemocyte count (THC)
The THC for each individual was determined immediately after sam-
pling using undiluted haemolymph in a Neubauer hemocytometer
(Shields et al., 1997).
2.2.2. Phagocytic rate (PR)
A slide-based phagocytosis method (Chen et al., 2005) was used
with modifications. Immediately after extraction from the pedal sinus
40 μl of fresh undiluted haemolymph was placed onto two Poly-L-
Lysine® glass slides. After incubating for 10 min, to allow adherence of
haemocytes, excess haemolymph was poured off and 40 μl of a zymosan
solution (108 particles ml−1 Zymosan A from Saccharomyces cerevisiae
(SIGMA catalogue #Z4250) suspended in sterile filtered seawater)
was added to the haemolymph and the slides were reincubated for
30 min. After 30 min, the slides were fixed with seawater formalin for
at least 20 min (10% formaldehyde in filtered seawater) and stained
with May Grunwald Giemsa. Haemocytes were counted in each of the
two smears (100 per slide) and the mean was taken. The percentage
phagocytosis was determined as [(phagocytic haemocytes) ∕ (total
adhered haemocytes)] ∗ 100.
2.2.3. MTS antibacterial assay
The bacterial culture used was a pathogenic strain of Vibrio harveyi
(sourced from the Fish Aquatic Health Unit, Department of Primary
Industries, Tasmania and maintained at − 80 °C in 10% glycerol).
V. harveyi colonies growing on nutrient agar were inoculated into
McCartney bottles containing nutrient broth and incubated overnight
at 37 °C on an orbital shaker (Ratek) at 200 rpm. The cultures were
returned to the exponential growth phase prior to antimicrobial assays
(test concentration ~ 108 bacteria per ml). The MTS assay was used to
measure the antibacterial activity present in the cell free haemolymph
against pathogenic bacteria. It is based on the reduction of the MTS
tetrazolium compound to a red formazan product by dehydrogenase
enzymes in metabolically active bacteria (Valkalia and Benkendorff,
2005). The procedure used followed Li et al. (2007). In brief, thawed
haemolymph was centrifuged at 500 ×g for 5 min to separate the cell
debris. Three 90 μl aliquots of supernatant were pipetted into a 96
well plate, before adding 10 μl of the V. harveyi culture into each well.
Negative controls consisted of 90 μl haemolymph added to 10 μl nutri-
ent broth. Positive controls consisted of 10 μl of the V. harveyi added
to 90 μl of the nutrient broth. After 30 min incubation, 20 μl of MTS
CellTitre 96® Aqueous One Solution (Promega) was added to each
well, then the plates were returned to the incubator (37 °C) for 2 h or
until development of the red formazan product in control wells. The
absorbance was measured at 492 nm using a 96 well plate reader
(Spectra Max 250). The background absorbance from the haemolymph
broth control was subtracted from the treatment wells. Bacterial
viability was calculated as a percentage of the absorbance (abs.) in pos-
itive control cultures, where the % antibacterial activity = 100 − ([abs.
sample ∕ abs. positive control] × 100).
2.2.4. Neutral red retention assay (NRR assay)
The NRR assay was modified from methods used by Harding et al.
(2004). The stock solution was composed of 2.28 mg of neutral red
powder dissolved in 1 ml of dimethyl sulphoxide (DMSO). The working
solution was 20 μl of the stock solution added to 1 ml of filtered
sterilised seawater. Fresh undiluted haemolymph (40 μl) was placed
on a Poly-L-lysine slide. The slide was incubated for 10 min to allow
cell adhesion, then excess fluid was tipped off and 40 μl of the neutral
red working solution was added. The slides were then coverslipped
and examined at 400 × magnification every 15 min until 50% of the
haemocytes had lysosomes significantly swollen greater than 7–8
times the original size or dye had leaked into the cytosol. At this time
point, the assay was halted and the previous time recorded was taken
as the NRR time. At each time point, at least 50 haemocytes were count-
ed over 3–4 fields of vision.
2.2.5. Biochemistry
Haemolymph samples were collected into eppendorf tubes, spun
down in a centrifuge (15 min at 13 ×g) and the cell free plasma stored
at − 80 °C until run on a biochemistry multichannel analyser (Roche
Modular SW version 7.5) at Gribbles Veterinary Pathology, Melbourne.
Calcium and phosphorus were run on serum settings. Chloride, potassi-
um, sodium and magnesium were run on urine settings. The within-
laboratory precision (coefficient of variation) for each variable was as
follows: Serum settings: calcium, 1.5%; and phosphorus, 2.7%. For
urine settings: chloride 5.6%; potassium 6.3%; sodium 5.5%; calcium 7%
and magnesium 7.3%. The choice of urine versus serum settings on the
equipment was based on the concentration of the ions in the abalone
haemolymph, which is markedly different to non-marine species.
2.2.6. Protein determination
Haemolymph was centrifuged (10 ×g, 10 min, 4 °C) and 10 μl of
haemolymph plasma was transferred in duplicate into 96-well micro-
plates (Flat Bottom Microtiter Plates, PathTech) to which 200 μl of dye
reagent (BCA protein assay reaction; Bicinchoninic Acid Kit, Sigma)
was added. Samples were then incubated at 37 °C for 1 h and absor-
bance was measured spectrophotometrically using a Multiskan Ex
microplate reader (Thermo Electron Corporation) at 595 nm. The
optical densities of the haemolymph plasma samples were compared
to a standard curve of bovine serum albumin (Sigma A7906) and results
were expressed as mg of protein·ml−1.
2.2.7. Leucine aminopeptidase assay (LAP)
Haemolymph plasma (100 μl) was transferred in duplicate to
96-well micro-plates. An aliquot of 75 μl of TrisHCl (0.2 M, pH 8.0)
followed by 25 μl of the substrate leucine p-nitroanilide (SIGMA
L2158, 10 mM in TrisHCl, 0.2 M, pH 8.0) was added to each well. The
plate was rapidly mixed for 10 s before each plate reading and absor-
bance was monitored at four minute intervals for 20 min at 405 nm
using a Multiskan Ex microplate reader. Negative controls without
haemolymph, but containing leucine p-nitroanilide substrate and
TrisHCl buffer were run in parallel and the values were subtracted
from test values. Enzyme activity was measured as the change in
absorbance·min−1·mg−1 of protein.
2.2.8. Phenoloxidase assay (DOPA)
Phenoloxidase activity was measured spectrophotometrically
by recording the formation of dopachrome produced from L -
dihydroxyphenylalanine (L -DOPA). Haemolymph plasma (100 μl)
was transferred in duplicate to 96-well micro-plates and 100 μl of
L-DOPA (30 mM L-3,4-dihydrophenylalanine, Sigma D9628, in HCl
0.2 M, pH 8) was added to each well. The microplate was rapidly mixed
for 10 s before each plate reading. The absorbance at 492 nm was record-
ed every 5 min at 20 °C over 30 min, using a Multiskan Ex microplate
reader. Negative controls, without haemolymph, but containing L-DOPA
and TrisHCl buffer, were run in parallel and the values subtracted from
test values to correct for possible auto-oxidation of the L-DOPA. Enzyme
activity was measured as the change in absorbance·min−1·mg−1 of
protein.
2.2.9. Acid phosphatase (ACP) activity
Haemolymph plasma (20 μl) was transferred in duplicate to 96-well
micro-plates. This was followed by the addition of 20 μl of sodium
acetate buffer (0.1 M pH 4.5, Sigma S8625) and either 40 μl of the
 C. Hooper et al. / Aquaculture 432 (2014) 26–37 29

substrate p-nitrophenyl phosphate disodium hexahydrate (5 mg pNPP, A

Sigma N4645, into 1 ml sodium acetate buffer) to test for ACP activity, or
40 μl of sodium acetate buffer (control to measure the background 2

absorbance). As a further control to measure any absorbance of the

substrate, inhibitor rows contained 20 μl haemolymph, 40 μl of the
substrate and 20 μl of an inhibitor (230 mg sodium tartrate dihydrate,
Ajax Firechem A 513-500G, in 1 ml of sodium acetate). The microplate
was incubated for 30 min at 37 °C in darkness prior to being spectro-
1
photometrically measured at 405 nm using a Multiskan Ex microplate
reader. p-Nitrophenol was used at a standard range of concentrations
to calibrate the method (Sigma 1048; 80 μl of 0 to 80 mM per well).
The activity was defined as the amount of phenol produced per mg
protein. Results are expressed as 10−3 μmol·mg−1 protein.

2.3. Histopathology 0

After weighing and taking haemolymph samples, the abalone were

euthanized by complete transverse section with a scalpel blade just B
below the level of the cerebral ganglia. Soft tissues were removed
from the shell and all viscera, as well as a transverse section of the
adductor muscle (foot muscle), and were placed into 10% seawater
formalin to fix for at least 24 h. Sections of viscera (left gill, heart, left
and right kidney, digestive gland, stomach and intestines), mouth
parts and foot muscle were placed into plastic cassettes and processed
routinely by the histopathology department in Gribbles Pathology to
make 4 μm thick paraffin embedded tissue sections, which were stained
with haematoxylin and eosin and cover-slipped.

2.3.1. Lesion scoring protocol

A range of tissues in a subset of abalone (3 abalone from each
treatment group each day) was examined under light microscopy
for differences between the heat-treated and control groups. Differ-
ences were seen in the left gills and digestive glands. A subset of
eight abalone per treatment group was then read blind to quantify
these changes and determine if the differences seen were statistical-
ly significant. As the variation between replicate abalone was high,
reader errors are not a relevant issue, and blind reading ensured no
bias between treatments.
Gill changes were scored by light microscopy at 400× and digestive
gland changes were scored at 200× under a light microscope (Olympus
BX45) using a subjective 0–5 score as outlined below (0 least affect, 5
most affected) or measured with a graticule (Olympus NE11A Ø24 mm
FIN-30593). The graticule was calibrated with an Olympus objective
micrometer containing a 0.01 mm grid.
2.3.1.1. Gills. The variations seen in the gill were the level of eosinophilia
in the sinus fluid (considered to reflect protein level and/or haem-
olymph pooling), goblet cell numbers at the tips of the leaflets and
areas of epithelial damage on the leaflets. The left gill was sampled in
each abalone and only gill leaflets sectioned at the level of corrugation
and containing the chitinous skeleton in the tip were examined, to
ensure that a similar area of each gill was being investigated. Fifty
leaflets were counted, with approximately 25 leaflets being examined
from each side of the gill. The examination started near the base of the
gill at approximately the 5th gill leaflet and every 3rd subsequent gill
leaflet was examined.
A subjective measure of 0–5 was given for the level of eosinophil-
ia of the fluid in the gill sinuses. If the fluid colour varied in different
parts of the gill, then the score was based on the deepest colour
(Fig. 1a, b).
Fig. 1. Gill leaflets showing haemolymph protein scores (eosinophilia) indicated by num-
bers and foci of epithelial necrosis N 25 μm (black arrows). a. Gill leaflets showing
haemolymph protein scores of 0, 1 and 2. The black arrows show foci of epithelial necrosis
N 25 μm. Goblet cells are marked by black arrow heads. Bar = 30 μm. Magnification ×400.
b. Gill leaflets showing haemolymph protein scores of 3 and 5. The black arrow shows a
focus of epithelial necrosis N 25 μm. Bar = 30 μm. Magnification ×400.
5 = very marked eosinophilia (dark red) or clumping of the protein-
aceous material
The number of goblet cells on the tips of 50 individual gill leaflets
(Fig. 1a) was counted and the mean was taken. The length of epithelial
damage on each of 50 gill leaflets was measured with the graticule.
Absence of epithelial cells, rupture of the gill leaflet with exudation of
fluid and sloughed epithelial cells were all considered forms of damage
(Fig. 1a, b). Any leaflet with N25 μm of epithelial damage on a leaflet was
given a score of 1 versus a score of 0 for leaflets with b25 μm of damage
or no damage. The mean of 50 leaflets was taken as the epithelial
damage score for that abalone.
2.3.1.2. Digestive gland. The section of digestive gland examined was a
1 cm long segment taken perpendicular to the left lateral surface of
the abalone and just caudal to the heart. Heat stressed abalone had
increased space in the interstitial tissue of the gland, considered to be
edema (pers. comm. Anna Mouton and Judith Handlinger), and
haemocyte infiltration (Fig. 2a, b).

0 = gill sinuses with no apparent fluid material

2.3.2. Digestive gland interstitial edema
1 = fluid that had minimal colour Interstitial edema was examined over 20 fields at 200× magnifica-
2 = mild eosinophilia (pale pink) tion in each abalone. Edema was measured both within the interstitium
3 = moderate eosinophilia (medium pink) surrounding the individual digestive gland acini (internal edema: 10
4 = marked eosinophilia (red) fields, Fig. 2a, b), as well as at the periphery of the digestive gland
30 C. Hooper et al. / Aquaculture 432 (2014) 26–37
A 0 = no edema
B fields with a 200× objective. Two measures were taken. One measure
C 2 = 10–50 haemocytes in a focus
Fig. 2. Photomicrographs of the internal area of the digestive gland. a. An internal area of
digestive gland showing multifocal edema and haemocyte infiltration. Black arrows show
foci of edema and arrow heads show foci containing haemocytes. Bar = 90 μm. Magnifica-
tion ×100. b. An internal area of digestive gland showing minimal edema (arrow) and no
haemocyte infiltration. Bar = 50 μm. Magnification × 200. c. The peripheral area of a
digestive gland (arrow head shows capsule) with extensive edema and haemocyte
infiltration separating the acini of the gland (2 black arrows point at acini). Greater than
50% of the interstitial tissue is edematous. Bar = 50 μm. Magnification ×200.
between the basement membrane of the gland and the capsule (periph-
eral edema: 10 fields, Fig. 2c).
In both the internal and external sites two measurements were
taken in each histological field to assess edema. The first measurement
was a subjective measure of the amount of the interstitial tissue
containing edema and a score of 0–3 was given. A mean of these scores
was taken over the 10 internal or external fields.
1 = b20% of the interstitium affected
2 = 20–50% of the interstitium was affected
3 = N50% of the interstitium affected
The second edema measure was the length in μm of the 3 longest
distances between the basement membranes of adjacent lobes in
each of the 10 fields examined (range seen was 0–45 μm). A mean
of the 30 measurements over 10 fields was taken. This mean was
multiplied by the mean of the subjective edema score above to
produce a score for the internal or external sites of the abalone. The
internal and external scores were then added for the total edema
score of that abalone.
2.3.3. Digestive gland haemocyte infiltration
Haemocyte infiltration in the interstitial tissue was measured over 10
was a score (0–3) for the number of foci containing haemocytes. A
mean of these 10 scores was taken.
0 = no foci with more than 5 haemocytes present
1 = b5 foci with at least 5 haemocytes infiltrated
2 = 5–10 foci with at least 5 haemocytes infiltrated
3 = N10 foci with at least 5 haemocytes infiltrated
The second measure gave a score (0–6) for the number of
haemocytes in the 3 foci with the highest cell concentrations. A mean
of these 30 scores was taken.
0 = b5 haemocytes in a focus
1 = b10 haemocytes in a focus
3 = 50–100 haemocytes in a focus
4 = 100–200 haemocytes in a focus
5 = 200–300 haemocytes in a focus
6 = N300 haemocytes in a focus
The mean of these 30 scores was multiplied by the mean of the 10
subjective haemocyte infiltration scores above. This total score is the
haemocyte density score for the digestive gland for each abalone.
2.4. Statistical analysis
Box plots were examined to assess the variances and look for outliers
and data was log or arcsin transformed when needed, to satisfy assump-
tions of variance homogeneity. Arcsin transformation was used for
parameters where any of the percent values were less than 20% or
greater than 80% (only PR data required transformation). Repeated
measures ANOVAs were performed using the Systat 12 software
package. Means for the abalone within each tank were calculated prior
to analysing the data, as the tanks are repeatedly sampled replicates,
while abalone are sacrificed sub-samples. For each of the measured
variables, we first used planned comparisons to determine whether
the measured changes during the experiment differed between the
heated abalone and the controls. These interaction comparisons calcu-
lated whether the changes between days 1 and 2 and between days 2
and 7 in the heat-treated abalone were significant, when compared to
changes over the same time in the control group. The next question
was whether the heat treatment had an overall effect. If there were
significant day effects, these were examined with post hoc tests, using
the Dunn–Sidak adjustment to ensure an experimentwise error rate of
0.05. A significance level of p = 0.05 was used for each test.
 C. Hooper et al. / Aquaculture 432 (2014) 26–37 31

3. Results 3.3. Phagocytic rate (PR)

3.1. Behaviour
As the temperature started to rise on day 1, the abalone in the
heat-treated tanks defecated. Within the first few hours of temperature
elevation, some of the heat-treated abalone elevated the shell several
millimetres above its resting level and these abalone repeatedly
swivelled on the axis of the pedal muscle. This swivelling ceased within
a few hours. The heat-treated abalone were as closely adhered to the
tank wall as those in control tanks throughout the experiment. When
removed from the tanks for sampling, the heat-treated abalone
remained still, without the pedal muscle extension (the righting reflex),
which was typically seen in the control animals.
3.2. Total haemocyte counts (THC)
The density of haemocytes was significantly elevated in the heat-
treated abalone relative to the controls across all three days of sampling
(Fig. 3a, Table 1) so that the overall effect of heat treatment was signifi-
cant, but the planned comparisons which showed changes over time for
the heated and control abalone did not differ significantly. The overall
change between days was significant and the post hoc tests showed a
significant drop in haemocyte numbers occurred between days 1 and 7
(p = 0.023).
The phagocytic rate of abalone haemocytes increased over time in
the controls, but not in the heat stressed abalone, (Fig. 3b). The compar-
isons showed that the changes between days 1 and 2 in the heat-treated
abalone were significant when compared to control abalone over the
same time period, and the overall change between days was also signif-
icant (Table 1). Post-hoc tests showed that the change between days 2
and day 7 was significant (p = 0.047).
3.4. Antibacterial assay
Antibacterial activity was significantly lower across all days in the
heated groups than in the control abalone, and the comparisons showed
no significant differences in how these two groups changed between
days (Fig. 3c, Table 1). The overall change between days was significant,
but the post-hoc comparisons indicated that only the difference
between days 1 and 7 was significant (p = 0.021).
3.5. Neutral red retention assay (NRR assay)
NRR times were significantly lower in the heated groups compared
to the control abalone, with no significant differences in the way these
groups changed between days (Fig. 3d, Table 1).

Fig. 3. Immune parameters in heat-treated and control abalone (mean ± SE). N = 12 in both treated and control groups. a. Log THC (cells × 106/ml ± SE). b. Arcsin phagocytic rate. c.
Antibacterial activity. d. NRR assay; the time in minutes from when the abalone haemolymph was added to the smears and lysosomal swelling occurred.
32
Table 1
Repeated measures ANOVAs of tank means for immune and enzyme assays. Abalone were
examined on days 1, 2 and 7 after tanks were heated from 16 °C to 26 °C (heat-treated) or
left at the ambient temperature of 16 °C (Control). N = 6 tanks in each group.
Parameter/effect df Mean square
Log THC
Heat effect 1 0.175
Between tanks error 10 0.020
Between days 2 0.087
D1–D2 control vs Heated 1 0.062
D2–D7 control vs Heated 1 0.011
Within tanks error 20 0.017
Arcsin PR
Heat effect 1 0.011
Between tanks error 10 0.017
Between days 2 0.063
D1–D2 control vs Heated 1 0.069
D2–D7 control vs Heated 1 0.003
Within tanks error 20 0.008
Antibacterial activity
Heat effect 1 1861.624
Between tanks error 10 75.668
Between days 2 477.263
D1–D2 control vs Heated 1 260.506
D2–D7 control vs Heated 1 76.677
Within tanks error 20 88.177
NRR
Heat effect 1 11,736.111
Between tanks error 10 898.819
Between days 2 2950.694
D1–D2 control vs Heated 1 66.667
D2–D7 control vs Heated 1 0
Within tanks error 20 916.528
Log LAP
Heat effect 1 0.840
Between tanks error 10 0.286
Between days 2 0.072
D1–D2 control vs Heated 1 0.555
D2–D7 control vs Heated 1 0.0054
Within tanks error 20 0.285
Phenoloxidase
Heat effect 1 0.008
Between tanks error 10 0.001
Between days 2 0.008
D1–D2 control vs Heated 1 0.0001
D2–D7 control vs Heated 1 0.0023
Within tanks error 20 0.001
Log ACP
Heat effect 1 0.006
Between tanks error 10 0.005
Between days 2 0.005
D1–D2 control vs Heated 1 0.0039
D2–D7 control vs Heated 1 0.0269
Within tanks error 20 0.017
Weight
Heat effect 1 65.961
Between tanks error 10 24.488
Between days 2 104.220
D1–D2 control vs Heated 1 26.18
D2–D7 control vs Heated 1 5.208
Within tanks error 20 35.136
a
Significant at p = 0.05 (within tanks effects Greenhouse–Geisser adjusted).
3.6. Leucine aminopeptidase activity (LAP)
The overall effect of heat treatment was not significant (Table 1),
although LAP tended to be higher in the heated than in the control
abalone on the first two days. Despite a progressive increase in LAP in
the control tanks between days 1 and 7 (Fig. 4a), neither the planned
comparisons nor the overall change between days was significant.
C. Hooper et al. / Aquaculture 432 (2014) 26–37
3.7. Phenoloxidase (PO) activity
Phenoloxidase activity increased over time in both controls and
heated abalone, leading to a significant effect between days (Fig. 4b,
F-ratio Table 1). It was also significantly lower overall in heat stressed abalone.
The day effect was due to significant differences between day 1 and 2
8.693a (p =0.011) and between day 1 and 7 (p = 0.014).
5.182a
3.672 3.8. Acid phosphatase (ACP) activity
0.637
ACP activity was marginally higher in heat treated abalone on day 7
(Fig. 4c), but there was no significant effect of any factor on log ACP in
0.668 this experiment (Table 1).
8.324a
8.587a
3.9. Weight
0.363
The mean weight of abalone did not differ significantly between heat
stressed and control abalone (Fig. 4d, Table 1), nor over time, despite an
24.603a apparent decline over the first two days.
5.413a
3.10. Biochemistry
2.954
0.870
Throughout the experiment, the mean value for all haemolymph ion
concentrations in heat-treated abalone mostly remained within the
13.057a range of the controls (Table 2), with no significant effect of treatment
or interactions between day and treatment for any of the haemolymph
3.219 ions or protein (p N 0.5). A significant day effect for Log Na (F2,55 =
0.073 3.874, p = 0.027), and Log Mg (F2,53 = 213.324, p b 0.001) was seen.
0
Tukey's post hoc test for days showed that days 1 to 2, days 2 to 7 and
days 1 to 7 were significantly different for both Log Na and Log Mg.
2.932
3.11. Histological changes in the gill
0.253
1.946 3.11.1. Gill sinus protein score
0.019 Heat had no significant effect on the protein level in the gill sinuses
(Fig. 5a, Table 3).
5.364a
3.11.2. Goblet cell numbers
9.445a The comparisons showed that the decline from days 1 to 2 and the
0.153 recovery from days 2 to 7 were both significant compared to the chang-
2.261 es in control abalone (Fig. 5b, Table 3). In addition there was a signifi-
cant overall change between days, due to the significant decline
between day 1 and day 2 (p = 0.016).
1.364
3.11.3. Epithelial damage in the gills
0.275
0.230 Heat had an overall effect on epithelial damage in the gill: the heat-
1.584 treated abalone had more gill leaflets with N 25 μm loss of epithelium
relative to the controls across all three days of sampling (Fig. 5c,
Table 3). The control abalone also had some leaflets with N 25 μm of
2.694 damage. The epithelial loss on the gill leaflets was multifocal rather
than diffuse, but one common focus of damage was the epithelium
2.966 just proximal to the ciliated columnar epithelium near the tips of the
0.745
0.148
gill leaflets (Fig. 1a, b).
3.12. Histological changes in the digestive gland
3.12.1. Interstitial edema
Although heat treated abalone appeared to have more edema on
days 2 and 7 (Fig. 6a) there was no significant overall heat effect, or
change over time (Table 3).
3.12.2. Haemocyte infiltration
Heat had a significant overall effect (Fig. 6b. Table 3). There was
greater infiltration of haemocytes in the digestive glands in the heat-
treated abalone relative to the control abalone.
 C. Hooper et al. / Aquaculture 432 (2014) 26–37 33

A 2
B 0.3

0.2

Phenoloxidase
1
Log LAP

0 0.1

Treatment
Control Treatment
Heat treated Control
-1 0.0 Heat treated
1 2 7 1 2 7
Days Days

C 2.0 D 80

70

1.5
60
Log ACP

Weight

50
1.0

Treatment 40 Treatment
Control Control
Heat treated Heat treated
0.5 30
1 2 7 1 2 7
Days Days

Fig. 4. Enzyme activity and weight in heat-treated and control abalone (mean ± SE). N = 12 in both treated and control groups. a. Log LAP activity (sigma units ml−1 mg−1 protein) in
haemolymph. b. Phenoloxidase activity in haemolymph (U mg−1 protein). c. Log ACP activity (μmol 10−3 mg−1 protein). d. Weight (g) of abalone at each sampling time.

4. Discussion farms in summer. Background microscopic lesions were present in the

These results demonstrate that severe heat stress in abalone causes
changes in behaviour, morphological changes within some tissues and
changes in haemolymph parameters. The observed changes occurred
at different times. Total haemocyte count was elevated in the heat
stressed abalone throughout the entire experiment, whereas the phago-
cytic rate was elevated on the initial day and then recovered. Other
parameters such as antibacterial activity and neutral red retention
time were depressed during the entire experiment. Histological changes
also varied temporally. Damage to the gill epithelium was seen in the
heat stressed abalone for the entire experiment, whereas haemocyte
infiltration into the digestive gland increased over the week. The results
help explain the increased incidence of infectious disease on abalone
control abalone and were distinguished from the heat treated group by
the degree of change using scoring methods to quantitatively measure
the change.
Behaviour can be a useful qualitative indicator of stress in abalone.
The elevation and swivelling of the shells of abalone on their pedal
muscles, with the loss of the righting reflex that were seen in the
heat-treated abalone in this study had some similarities to those
noted in abalone infected with abalone viral ganglioneuritis virus
(Hooper et al., 2007b) and animals from tanks to which anaesthesia
had been added (Burke et al., 2001; Hooper et al., 2011). Why severe
heat leads to a decreased righting reflex is not clear. A slowing or loss
of the righting reflex has also been noted in abalone injected with
tetracycline (R.W. Day et al., 1995; Pirker and Schiel, 1993) and could

Table 2
Mean ± SE of haemolymph ions and protein from abalone that were sampled on days 1, 2 and 7 after tanks were heated from 16 °C to 26 °C (Heated) or left at the ambient temperature
(Control). N = 12 in each treatment group for each day.

Day 1 Day 2 Day 7

Heated Control Heated Control Heated Control

CL mmol/L 501 (10) 509 (16) 518 (6) 510 (5) 526 (14) 521 (6)
K mmol/L 10.3 (0.2) 10.1 (0.2) 10.7 (0.2) 10.4 (0.1) 10.4 (0.3) 10.2 (0.2)
Na mmol/L 400 (6) 396 (12) 408 (5) 402 (3) 410 (10) 413 (4)
Ca mmol/L 9.5 (0.1) 9.4 (0.2) 9.9 (0.1) 9.7 (0.1) 9.8 (0.2) 9.8 (0.1)
Mg mmol/L 33.1 (0.2) 33.2 (0.1) 32.7 (0.1) 32.5 (0.1) 34.6 (0.4) 34.7 (0.2)
PO4 mmol/L 0.5 (0.1) 0.4 (0.1) 0.1 (0.03) 0.4 (0.1) 0.2 (0.03) 0.2 (0.1)
Protein g/L 9.1 (0.6) 9.8 (0.6) 10.1 (0.2) 10.7 (0.2) 8.8 (0.1) 8.6 (0.3)
34
A B
0.7
0.6
Log gill sinus protein score
0.5
0.4
0.3
0.2
0.1
1 2
Days
C
0.3
Log gill epithelial damage score
0.2
0.1
0.0
1 2
Days
Fig. 5. Gill histopathology scores (mean ± SE). a. Log gill sinus protein score. b. Log gill goblet cell mean. c. Log gill epithelial damage score.
be a general stress effect. The elevation and swivelling of the shell have
similarities to the behaviour exhibited in attempting escape from
predators (R. Day et al., 1995; Parsons and Macmillan, 1979). It is likely
that abalone have only a limited repertoire of behavioural stress
responses with similar behaviour occurring with varied stressors and
illnesses.
Some haemolymph parameters, such as the PR% and THC showed
changes soon after the water temperature was raised (on day 1), but
declined to near the level of the controls by day 2, suggesting not only
an acute stress response but also early acclimatization. Haemocyte
counts were significantly elevated by heat and the greatest difference
was seen on day 1, similar to the observations of Dang et al. (2012)
and Cheng et al. (2004a). Transient haemocytosis has been correlated
with elevation in stress hormones in abalone (Malham et al., 2003;
Ottaviani and Franceschi, 1997). Stress leukocytosis in vertebrates can
be due to cell mobilization or increased myelopoiesis (Engler et al.,
2004a, 2004b; Gray et al., 1989; Maxwell, 1993), with the former
more likely to explain the haemocytosis we observed on day 1.
The histological changes in the digestive gland showed a trend to
progressively worsen over the week, indicating that prolonged high
temperature could have an increasingly deleterious effect. It also
suggests that the greater haemocytosis on day 1 is due to mobilization
of haemocytes from tissues into the haemolymph due to acute heat
stress, followed by infiltration into tissues such as the digestive gland
by day 7. Thus immunological assessment of haemolymph THC appears
to correlate with the observed histological changes.
The phagocytic rate in this study rose on day 1 but declined to a level
similar to that of control abalone by day 2, suggesting that the elevated
C. Hooper et al. / Aquaculture 432 (2014) 26–37
0.9
0.8
Log gill goblet cell mean
0.7
0.6
0.5
0.4
Treatment Treatment
0.3
Control Control
Heat treated Heat treated
0.2
7 1 2 7
Days
Treatment
Control
Heat treated
7
heat had an acute effect but the cells were able to rapidly acclimatize.
The elevation in phagocytic rate on the first day of the experiment
was unexpected because previous research into temperature stress
showed a decline in phagocytic rate of heat stressed abalone (Cheng
et al., 2004a; Travers et al., 2008). The absence of a depressed phagocytic
rate in this experiment suggests that this component of the immune
response is more resistant to temperature elevation compared to
other components, such as antibacterial activity. The reasons for the
initial increased phagocytic rate on day 1 could include immune stimu-
lation accompanying the elevated haemocyte count or it may possibly
reflect increased metabolic activity, which has been reported with
elevated temperature (Vosloo and Vosloo, 2010).
In contrast to the apparent acclimatization in some of the immune
parameters, other haemolymph parameters indicate an apparent lack
of acclimatization to heat stress in abalone. Antibacterial activity and
NRR times were consistently lower in the heat-treated group over
each day tested, suggesting a lack of acclimatization to the continued
elevated temperature. Similar effects of heat stress on antibacterial
activity have been reported for abalone (H. rubra) in both laboratory
and field experiments (Dang et al., 2012). Similar effects of heat stress
were also observed on antibacterial activity in Pacific Oyster (Crassostrea
gigas) haemolymph (Li et al., 2007). This may be due to diminishing
stores of antimicrobial factors without replacement during prolonged
stress due to energy diversion away from non-essential processes.
Phenoloxidase activity has been identified as another haemolymph
parameter that is negatively impacted in heat stressed abalone (Cheng
et al., 2004a; Travers et al., 2009). Our finding of significantly lower
phenoloxidase activity in heat stressed abalone relative to controls is
 C. Hooper et al. / Aquaculture 432 (2014) 26–37 35

Table 3 antimicrobial intermediate compounds (Cerenius et al., 2010) and

Repeated measures ANOVAs of tank means for log histopathology scores. Abalone from 5 thus a decline in phenoloxidase activity may have contributed to the
control tanks and 4 heated tanks were examined on days 1, 2 and 7.
simultaneous decrease observed in antibacterial activity.
Parameter/effect df Mean squares F-ratio Haemolymph electrolytes were not affected by increased tempera-
Log gill protein score ture, indicating that abalone are able to maintain ionic homeostasis
Heat effect 1 0.001 0.569 under this degree of heat stress. Molluscan ionic status can adapt quick-
Between tanks error 7 0.002 ly to regular changes (Byrne and McMahon, 1991). Any perturbations
Between days 2 0.015 1.482
that may have occurred in the ions due to heat stress must have
D1–D2 control vs Heated 1 0.00001 0.001
D2–D7 control vs Heated 1 0.0003 0.027 resolved in less than the 5 h between heat stress commencing and the
Within tanks error 14 0.010 first samples being taken. Further work monitoring the haemolymph
ions during the first few hours after a severe stress would be useful to
Log goblet cell numbers
Heat effect 1 0.018 3.966 confirm this hypothesis.
Between tanks error 7 0.004 The prolonged effects of severe heat stress in abalone are also
Between days 2 0.029 7.183a manifested in damage to tissue epithelium, which can be observed in
D1–D2 control vs Heated 1 0.020 4.997a histological sections. The results have shown that background damage
D2–D7 control vs Heated 1 0.035 8.632a
to gill epithelium exists, but that thermal stress causes increased
Within tanks error 14 0.004
damage that can be measured by quantitative histology. The epithelial
Log gill epithelial damage damage seen in this experiment was similar to that described by
Heat Effect 1 0.017 19.397a
Mouton (2003) as being associated with transport stress, but Mouton
Between tanks Error 7 0.001
Between days 2 0.001 0.650 did not quantify the changes as seen in this experiment. Mouton
D1–D2 control vs Heated 1 0.0003 0.350 (2003) speculated that transport stress may be due in part to the
D2–D7 control vs Heated 1 0.0004 0.424 increased temperatures experienced when out of the water. The gill
Within tanks Error 14 0.001
necrosis observed in the current experiment supports this conjecture.
Log dig. gland edema While the current experiment does not exclude other causes of epithe-
Heat effect 1 0.018 0.444 lial damage in gills, it does demonstrate that heat alone can cause this
Between tanks error 7 0.042
effect. However, since there are background levels of damage, quantify-
Between days 2 0.004 0.032
D1–D2 control vs Heated 1 0.193 1.548
ing the damage is needed to indicate serious heat stress. Mouton (2003)
D2–D7 control vs Heated 1 0.113 0.905 also referred to increased gill fluid accumulation with transport stress,
Within tanks error 14 0.125 which corresponds to the trend to increased proteinaceous fluid in the
Log dig. gland haemocytes
gills of heated abalone in our experiment. Mouton described the lesions
Heat effect 1 0.203 12.229a she saw but did not report any morphometric studies to confirm that
Between tanks error 7 0.017 the gill sinus fluid increased significantly or was only a trend, such as
Between days 2 0.000 0.015 we noted.
D1–D2 control vs Heated 1 0.001 0.075
The damaged gill epithelium is a portal of entry for bacteria. In
D2–D7 control vs Heated 1 0.044 3.388
Within tanks error 14 0.013 summer, bacteria can increase in both number and virulence, due to
a
upregulated bacterial gene expression of virulence factors such as
Significant at p = 0.05 (within tanks effects Greenhouse–Geisser adjusted).
adhesins (Parveen et al., 2008; Toren et al., 1998), which may explain
the increased incidence of bacterial disease reported by farmers during
consistent with these reports. Interestingly, Wang et al. (2004) found summer. There was no significant recovery in gill epithelia after 7 days,
that phenoloxidase activity did not change significantly in abalone which suggests that with the continued elevated temperature, the
injected with pathogenic bacteria, and speculated that phenoloxidase damaged gill epithelium was unable to be repaired. Epithelial damage
may not be important in the immune system of abalone. However the can be quickly repaired in some tissues. After incising the foot of abalo-
injection method used by Wang et al. (2004) may have produced a ne, the haemocytes can form a layer along the wound surface within
local response to injury rather than a systemic haemolymph response, 8 h; and by 4 days later can form layers 5–20 cells deep (Armstrong
as heat stress would provide. The phenoloxidase system produces et al., 1971). However, either the damage to gill epithelium associated

Fig. 6. Digestive gland scores taken from 20 fields per abalone (mean ± SE). a. Log interstitial edema score. b. Log haemocyte infiltration score.
36 C. Hooper et al. / Aquaculture 432 (2014) 26–37
with heat takes longer to heal, or the persistent elevated heat in this
study interfered with repair.
Anecdotally, abalone farm workers have commented on increased
mucus seen in the tank water after increased temperatures. In this
study, the goblet cells full of mucin in the heat-stressed group declined
on day 2, but had recovered by day 7. This could reflect release of mucin
as a response to heat stress, with subsequent recovery of mucin produc-
tion by these cells as they acclimatize to the heat. Increased mucus
secretion is likely to be a general stress response in gastropods, rather
than one associated with heat only (Noble et al., 2009; O'Omolo et al.,
2003).
Heat led to significantly increased infiltration of haemocytes into the
digestive gland. There was a trend to increased edema in the digestive
gland due to heat stress but it was not significant. Chronic husbandry
problems such as nutritional stress and overstocking have been report-
ed to lead to digestive gland edema and atrophy (Mouton, 2003).
Prolonged sublethal heat stress has been shown to lead to digestive
gland atrophy and replacement of the glandular tissue with fibrosis
(Braid et al., 2005), which is likely to be irreversible. Oxygen consump-
tion rises with increased temperatures, and organs with high metabolic
rates may suffer an inadequate supply earlier as temperatures rise (Van
Dijk et al., 1999). The digestive gland beta cells contain abundant
mitochondria (Bevelander, 1988) suggesting that a high metabolic
rate is present in this organ. Higher temperatures leading to elevated
metabolism will lead to elevated tissue catabolism and consumption
of energy stores (Vosloo and Vosloo, 2010). The high metabolic rate
suspected in the digestive gland may partially explain the lesions seen
in this organ developing within one week of heat stress. Further work
to characterize the degree and timing of the histologic changes follow-
ing severe thermal stress is needed.
The presence of similar changes in histological sections of both heat-
stressed and control groups shows that background changes are present
in farm stock and therefore the presence of these changes is not diagnos-
tic of a particular stress. The heat-stressed abalone had more severe
changes, which could be measured using the methods described here.
Extensive inter-abalone variation in the histology indicates that the
presence of changes in individual abalone or in low numbers of stock is
not diagnostic. Using morphometric techniques in experimental studies
of other stressors could help in analysing management problems.
Stress associated with summer will be more complex than simply
increases in temperature, including problems with water quality and
increased bacterial counts. Further work needs to identify which
components of the farm environment cause stress during summer,
and which stressors have the most effect both individually and in syner-
gistic combinations. Whether the changes seen histologically are due
only to heat or could occur with other environmental problems needs
to be investigated. Over summer, water temperature increases at
varying rates and stock are exposed to a higher temperature for varying
times. The rate of temperature increase to which abalone can fully
acclimatize is not known. A longer exposure to a slowly rising water
temperature can have a more detrimental effect on marine animals
than a faster rise in temperature over a short period (Cocking, 1959;
Mora and Maya, 2006). This could be due to increased loss of body
reserves over a prolonged time (Mora and Maya, 2006) or due to
prolonged exposure to stressful water temperatures even when these
are non-lethal (Cocking, 1959). A smaller temperature spike preceding
a severe elevation can have the effect of reducing mortality due to the
production of inducible heat shock proteins that provide protection
for the more severe heat shock (Li et al., 2007).
This study shows that severe prolonged elevated water temperature
will cause histological alteration in some tissues and many immune
parameters, but has no significant effect on the haemolymph electro-
lytes. Sublethal but severe heat stress leads to immunosuppression
and damage in the gills and digestive gland that will make abalone
more vulnerable to infection during the summer months. The results
show that sublethal indicators of immune stress based on haemolymph
assays occur concurrently with histological changes indicative of tissue
damage. The results also show the benefit of using quantitative histolo-
gy to differentiate background changes seen in tissues due to severe
heat stress.
Acknowledgements
We thank the Australian Government Fisheries Research and Devel-
opment Corporation (FRDC grant No. 2004/233), which supplied the
funding for this research; Great Southern Waters Limited for allowing
the research to be carried out and supporting it on their abalone farm;
Bruce Abaloz of the University of Melbourne and Jo Mercuri and Kelly
Saint at Gribbles Veterinary Pathology for processing the Histology;
Phuong Nguyen and Charan Walia of Gribbles Veterinary Pathology
for the biochemistry processing and John Ahern who runs the aquarium
in the Department of Zoology in the University of Melbourne and helped
me set up the tanks.
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