Journal of Fish Diseases 2012, 35, 563–568
doi:10.1111/j.1365-2761.2012.01397.x
Enhancement of Hsp70 synthesis protects common carp,
Cyprinus carpio L., against lethal ammonia toxicity
Y Y Sung1,2, R J Roberts3 and P Bossier4
1 Department of Aquaculture Science, Faculty of Fisheries and Aqua-Industry, Universiti Malaysia
Terengganu (UMT), Kuala Terengganu, Malaysia
2 Institute of Marine Biotechnology, Universiti Malaysia Terengganu (UMT), Kuala Terengganu, Malaysia
3 Hagerman Fish Culture Research Laboratory, Hagerman, ID, USA
4 Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience Engineering, Ghent University,
Belgium
Abstract
Exposure to TEX-OE, a patented extract of the
prickly pear cactus (Opuntia ficus indica) containing
chaperone-stimulating factor, was shown to protect
common carp, Cyprinus carpio L., fingerlings
against acute ammonia stress. Survival was enhanced twofold from 50% to 95% after exposure to
5.92 mg L)1 NH3, a level determined in the
ammonia challenge bioassay as the 1-h LD50 concentration for this species. Survival of TEX-OEpre-exposed fish was enhanced by 20% over
non-exposed controls during lethal ammonia challenge (14.21 mg L)1 NH3). Increase in the levels of
gill and muscle Hsp70 was evident in TEX-OEpre-exposed fish but not in the unexposed controls,
indicating that application of TEX-OE accelerated
carp endogenous Hsp70 synthesis during ammonia
perturbation. Protection against ammonia was
correlated with Hsp70 accretion.
Keywords: Ammonia stress, carp, Cyprinus carpio,
Heat shock proteins, TEX-OE.
Introduction
Prokaryotic and eukaryotic organisms, including
humans, all possess a group of highly conserved
proteins termed heat shock proteins (Hsps). As
molecular chaperones, Hsps bind the exposed
Correspondence Dr Y S Yeong, Department of Fisheries and
Aquaculture, Faculty of Agrotechnology and Food Science,
University Malaysia Terengganu (UMT), 21030, Kuala Terengganu, Malaysia (E-mail: [email protected])
2012
Blackwell Publishing Ltd
563
hydrophobic surfaces of non-native proteins introduced into the body (Robert 2003) and of those not
fully synthesized, folded, assembled or localized to
the appropriate cellular compartment (Feder &
Hoffman 1999; Mchaourab, Godar & Stewart
2009). They also maintain other proteins in
folding-competent, folded or unfolded states, contribute to the folding of nascent and altered
proteins, assist protein localization, import and/or
export between cell organelles and target non-native
or aggregated proteins for degradation and removal
from cells. They are also known as stress proteins
and are constitutively expressed, representing
5–10% of the total protein in healthy growing cells
and two or three times that amount when induced
by stressors such as heat, cold, nutritional deficiencies, oxygen deprivation and disease (Pockley 2003;
Pockley, Muthana & Calderwood 2008; Roberts
et al. 2010), all of which can affect protein
conformation. Upon up-regulation of Hsps, cells
are better able to adapt to gradual changes in their
environment and to tolerate lethal conditions.
In relation to aquatic organisms, the Hsp70
family of heat shock proteins is perhaps the most
widely studied. Generally, interest has centred on its
well-recognized role in enhancement of the thermal
resistance response to heat stress. Examples include
observations on Hsp70-induced thermotolerance
generated in coho salmon, Oncorhynchus kisutch
(Walbaum), as a result of a sublethal heat shock
(Arkush, Cherr & Clegg 2008), and elevated Hsp70
promoted resistance to lethal temperature of the
brine shrimp, Artemia franciscana (Kellogg) (Clegg
Journal of Fish Diseases 2012, 35, 563–568
et al. 2000; Sung et al. 2008). Molluscs such as
Pacific oyster, Crassostrea gigas (Thunberg) (Clegg
et al. 1998), and California native oyster, Ostreola
conchaphila (Carpenter) (Brown et al. 2004), are
able to survive temperatures up to 39 C, a level
well above their normal lethal temperature, when
they are primed initially with a sublethal heat shock
at 34 C. Hsps are also involved in cross-tolerance,
a phenomenon whereby aquatic organisms acquire
increased tolerance to physiological insult following
an initial transient, albeit different, stress, a capability frequently occurring concomitantly with Hsp
accretion (Sung et al. 2007). With respect to biotic
stress tolerance, Hsp70 has also been shown to
protect the brine shrimp (Sung et al. 2007, 2008,
2009a,b), the black tiger prawn, Penaeus monodon
(Fabricius) (de la Vega et al. 2006), and platyfish,
Xiphophorus maculatus (Günther) (Ryckaert et al.
2010), against pathogenic microbe challenge.
In most of the above-cited examples, heat shock
was used to enhance Hsp production. Whilst
effective, this can in itself cause significant mortalities
in aquatic organisms. A less traumatic means of
enhancement of Hsp production would therefore
have considerable practical value. Pro-Tex, the
aquatic form of TEX-OE, is a patented extract of
the prickly pear cactus (Opuntia ficus indica). It has
been shown to accelerate and elevate Hsp synthesis in
humans (Wiese et al. 2004) and fish (Sandilands,
Drynan & Roberts 2009; Roberts et al. 2010),
protecting them against a wide variety of stressors.
The current study was carried out to investigate
whether application of TEX-OE chaperone-stimulating factor could be shown to protect carp,
Cyprinus carpio L. against toxic ammonia levels, a
common cause of significant mortalities of fish in
aquaculture and in aquarium fish, particularly during
transportation. The correlation between enhanced
tolerance and Hsp70 expression in muscle and gill
during ammonia perturbation was determined.
Materials and methods
Preparation of Opuntia ficus indica extract
solution
Two microlitres of Pro-Tex [Bradan Limited, a
soluble variant of TEX-OE (Batch no. J3TexET08066)] for use in fish culture was diluted with
50 L of dechlorinated freshwater following the
manufacturerÕs recommendation, with the solution
mixed well by gentle shaking.
2012
Blackwell Publishing Ltd
564
Y Y Sung et al. Hsp70 and ammonia toxicity
Experimental animals and median lethal
ammonia (1-h LC50) determination
Carp juveniles measuring approximately 5 cm
(0.54 g) were acclimatized with constant aeration at
28 C in the hatchery of University Malaysia Terengganu (UMT) 7 days prior to experiment. During
acclimatization, fish were fed ad libitum on granular
pellets consisting 60% crude protein. Faeces were
removed, and 50% of the rearing water replaced daily
to maintain optimal water quality.
Short-term static toxicity tests without water
replacement were performed to estimate the acute
toxicity levels of ammonia under the experimental
conditions. Groups of 20 fish were transferred to a
2-L test aquaria 12 h prior to exposure. The water pH
and temperature were controlled to 7.0 and 28C,
respectively. Fish were then subjected to 1-h incubation in 2.37, 4.74, 9.48, 11.84, 14.21, 16.58 and
18.95 mg L)1 NH3, with ammonium chloride
stock solution added to each experimental aquarium
and adjusted to compensate for water salinity,
temperature and pH variation to reach the desired
ammonia concentrations (Alcaraz et al. 1999; Schuler et al. 2010). Unexposed animals served as the
controls. Aeration ceased during exposure to prevent
ammonia evaporation. Mortality was determined
after 1 h by counting dead animals, and the percentages calculated as Nt · 100/No, where Nt and No are
final and initial numbers of fish, respectively (Sung
et al. 2008). Morbid fish were considered dead. The
concentration that causes 50% mortalities within
1 h, obtained from the mortality curve, is considered
as the 1-h LD50. Lethal concentration refers to the
concentration that causes total mortalities (LCT).
Each bioassay was performed in triplicate.
Ammonia challenge test
To test whether TEX-OE confers enhanced
ammonia tolerance, two groups of fish (one preexposed for 2 h with TEX-OE by immersion and
another without TEX-OE immersion, each consisting of 20 fish) were challenged at LC50
(5.92 mg L)1) and LCT (14.21 mg L)1) NH3 for
1 h. Survivals were determined as above.
Protein extraction, SDS–PAGE and Western
immunoblotting
Fish that survived after challenge at median lethal
ammonia (LD50) concentration were killed by
pithing prior to removing muscle and gill tissues by
Journal of Fish Diseases 2012, 35, 563–568
dissection. Protein extraction was performed as
described by Sung et al. (2007, 2008). About
20 mg of wet tissue was rinsed several times with
sterile water prior to being homogenized in 100 lL
of cold buffer K (150 mm sorbitol, 70 mm potassium
gluconate, 5 mm MgCl2, 5 mm NaH2PO4, 40 mm
HEPES, pH 7.4) (Clegg et al. 2000) supplemented
with a protease inhibitor cocktail (Sigma-Aldrich
Inc.-P8340) as recommended by the manufacturer.
Aliquots of homogenate were combined with equal
volumes of 2 times sodium dodecyl sulphate (SDS)
sample buffer, vortexed, heated at 95 C for 5 min
(Laemmli 1970), cooled and centrifuged at 4000 rpm
for 10 min. Ten microlitres of protein samples was
loaded to the lane of 10% poly-acrylamide gels and
electrophoresed at 130 volts for 15 min and followed
by 150 volts for 45 min. HeLa cell lysate (HC) (heat
shocked, Bioreagent-LYC-HCL100) served as a
positive control. Two gels were run simultaneously:
one stained with Coomassie Biosafe (Bio-Rad Laboratories) and another transferred to a polyvinylidene
fluoride transfer membrane (Bio-Rad Immun-Blot
PVDF) for antibody probing. Membranes were
incubated in blocking buffer (50 mL of phosphatebuffered saline containing 0.2% (v/v) Tween-20 and
5% (w/v) bovine serum albumin) for 60 min at
25 C. Hsp70 monoclonal antibody (1:5000 dilution, Thermo Scientific MA3-006) was used to detect
both constitutive and inducible isoforms of the
Hsp70. Goat anti-mouse IgG F(abÕ)2 polyclonal
antibody, HRP (1:2000 dilution, Bioreagents-SAB100J), was used as secondary antibody. For detection,
0.7 mm diaminobenzidine tetrahydrochloride dihydrate (DAB) was used as a substrate in association with
0.01% (v/v) H2O2 in 0.1 m Tris-HCL (pH 7.6) (Sung
et al. 2007, 2008).
Y Y Sung et al. Hsp70 and ammonia toxicity
as the 1-h LD50. Total mortalities first occurred at
14.21 g L)1 NH3, and this concentration was
considered as the lethal ammonia concentration
(LCT) (Fig. 1).
Tex-OE enhances carp survival during acute
ammonia stress
Exposure to TEX-OE within the water column for
2 h before challenge significantly enhanced the
tolerance of the carp to acute ammonia challenge.
Survivals were boosted twofold from approximately
50% to 95% after exposure to the median LCT
(LD50) (Fig. 2). When fish exposed to TEX-OE
in the water column for 2 h were then exposed to
acute ammonia challenge at the 100% lethal
challenge level, the number of viable animals was
also enhanced, with survival of 20% of fish
challenged, whereas total mortality occurred in the
controls (Fig. 3).
Figure 1 Cumulative mortalities of Cyprinus carpio fingerlings
after challenge for 1 h in different ammonia (NH3) concentrations. Data presented are mean standard deviations. Experiments were repeated once.
Statistics
Survival percentage (%) were ArcSin to satisfy
normality and homocedasticity requirements whenever necessary. Significant differences were determined by performing one-way ANOVA with
software SPSS version 11.5 for Windows.
Results
One-hour median and lethal ammonia
concentration
NH3 at 5.92 g L)1 killed 50% of the animals
within 1 h; thus, this concentration was considered
2012
Blackwell Publishing Ltd
565
Figure 2 Survival of control carp and fish treated with TEXOE followed by exposure to 1-h LD50 (5.92 mg L)1 NH3).
Data presented are mean standard deviations. *Statistical
differences as against control treatment at P < 0.05. Experiments were repeated once, indicated as Exp 1 and Exp 2.
Y Y Sung et al. Hsp70 and ammonia toxicity
Journal of Fish Diseases 2012, 35, 563–568
Gill
Muscle
(a)
250 kDa
150 kDa
100 kDa
75 kDa
50 kDa
Figure 3 Survival of TEX-OE pre-exposed carp after challenge
with lethal ammonia concentration (LCT) (14.21 mg L)1 NH3).
Total mortality occurred in the unexposed controls. Data
presented are mean standard deviations. Experiments were
repeated once, indicated as Exp 1 and Exp 2.
37 kDa
M
C
T
C
T
Muscle
(b)
Gill
250 kDa
Tex-OE enhances muscle and gill Hsp70
The Coomassie-stained gels revealed induction of a
40-kDa protein, a 70-kDa protein and a 90-kDa
protein in the muscles of carp pre-exposed to TEXOE. A single protein band of approximately
70 kDa was enhanced in the gill tissues (Fig. 4A).
Hsp70 was detected in the HC, which served as
the positive control in this study, revealing a single
protein band at approximately 70 kDa. Muscle
Hsp70 was slightly enhanced in fish pre-exposed to
TEX-OE. Augmentation of Hsp70 was evident in
the gill tissues (Fig. 4B).
Discussion
Ammonia is extremely harmful to fish, with the
unionized form NH3, more toxic than NH4+.
Ammonia accumulation in the rearing water may
result from fish metabolic waste, assimilation
process from uneaten feed and nitrogen-containing
substances from agricultural effluent (Santhi et al.
1992). Total ammonia of more than 0.02 mg L)1
is not desired in aquaculture systems because it
reduces fish survival, inhibits growth and causes a
variety of physiological malfunctions (Israeli-Weinstein & Kimmel 1998). In this study, exposure to
5.92 mg L)1 NH3 for 1 h resulted in 50% mortalities, whereas 14.21 mg L)1 killed all experimental fish; thus, these concentrations were considered
as the 1-h LD50 and LCT, respectively, for
common carp under the experimental conditions
extant. Reduced swimming activities, increased
respiration as well as gill and tissue erosion occur,
suggesting that mortality is caused by gill damage, a
2012
Blackwell Publishing Ltd
566
150 kDa
100 kDa
75 kDa
50 kDa
M
HC
C
T
C
T
Figure 4 A The protein profiles of carp pre-exposed to TEXOE (T) and unexposed controls (C) during 1-h LD50 stress
(5.92 mg L)1). Muscle and gill tissues were collected, and the
extracted protein electrophoresed in 10% Coomassie-stained
SDS polyacrylamide gel. Box, elevation of p40, p70 and p90 in
muscles of fish exposed to TEX-OE. Arrow, induction of p70
in gills. M, molecular mass standards are on the left in
kilodaltons (kDa). B Immunodetection of muscle and gill
Hsp70. HC, HeLa cell lysates as a positive control.
typical sign of ammonia toxicity in many fish
species (Benli, Köksal & Özkul 2008).
Ammonia, particularly NH3, diffuses easily
across the gill membranes, causing severe gill
damage that disrupts respiratory functions (Russo
1985; Bhakta 2006). Exposing carp to TEX-OE,
however, enhanced tolerance towards the detrimental effect of ammonia, evidenced by a twofold
increase in survival after the median lethal concentration challenge. Remarkably, 20% of those that
were pre-exposed to TEX-OE also survived lethal
ammonia challenge, a situation whereby total
mortality of the experimental fish should occur.
This protection may be explained by the high
induction of gill and/or muscle Hsp70, outcomes
similar to other aquatic organisms encountering
ammonia stress (Roberts et al. 2010) or perturbation of a different nature (Brown et al. 1992; Sung
Y Y Sung et al. Hsp70 and ammonia toxicity
Journal of Fish Diseases 2012, 35, 563–568
et al. 2007, 2008). As one example, heat shock
followed by 4- to 48-h recovery enhances the ability
of tide pool sculpin to withstand osmotic and
hypoxic stress, with survival increasing from 68% to
96% and 47% to 76%, respectively. The strongest
association between increased Hsp70 and crosstolerance is observed in tide pool sculpin gills
(Todgham, Schulte & Iwama 2005).
The development of stress tolerance in fish
following the exposure to TEX-OE is correlated
with Hsps expression. This result is in agreement
with the findings of Camilleri (2002) and Roberts
et al. (2010), who reported that pretreatment with
TEX-OE via immersion induced angelfish, Pterophyllum scalare (Schultze), gill Hsp70 expression
and promoted a twofold survival increase when
exposed to 1.1 mg L)1 free ammonia, which was
the 24-h LC50 for this species. When TEX-OE
was used to prestimulate fish before exposure to
stressors, circulating Hsp levels were detectable after
the first sign of stress. Hsps were rapidly induced,
and the levels were higher than those normally
occurring in unexposed fish subsequent to a heat
stress (Balucci 2005; Roberts et al. 2010). The
mechanism by which Hsps protect against stress has
yet to be determined, but the increment of
intracellular level of Hsp70 may mediate tolerance
by preventing protein denaturation, refolding damaged proteins or ensuring degradation of irreversibly
damaged proteins, thus preventing accumulation of
abnormal proteins and their aggregates (Hartl &
Hayer-Hartl 2009; Mchaourab et al. 2009). These
processes are crucial in maintaining a normal
cellular homoeostasis during stress.
On a practical note, application of TEX-OE
might also induce Hsps other than Hsp70. As
revealed by SDS-PAGE, the expression of a 40- and
90-kDa protein was apparent in carp pre-exposed to
TEX-OE during ammonia perturbation. Further
studies are required to substantiate whether these
proteins represent Hsp40 and 90. It is noteworthy
that a multi-component chaperone complex is
required to establish stress tolerance, a case seen in
the thermotolerance acquisition of insects and
aquatic organisms (Duncan 2005). Whatever the
outcomes might be, the current investigation shows
that Pro-Tex, the form of TEX-OE used in the
study, induces Hsp70 and enhances carp tolerance
against the deleterious effects of ammonia. Its usage
is demonstrably a safe and effective means of
induction of rapid Hsp synthesis during stress, with
high levels of survival in circumstances where severe
2012
Blackwell Publishing Ltd
567
losses might be expected. The study suggests that
Hsps play a crucial role in ammonia stress tolerance
in carp.
Acknowledgements
This work was supported by the E-Science Fund
Project No. 05-01-12-SF1006 from the Ministry of
Agriculture (MOA), Malaysia to YYS. We thank Dr
Hii Yii Siang, Mr Chou Ching Chung and Mr
Chan Huan Hui for their assistance in the preparation of the manuscript.
References
Alcaraz G., Chiappa-Carrara X., And V.E. & Vanegas C. (1999)
Acute toxicity of ammonia and nitrite to white shrimp Penaeus
setiferus. Journal of the World Aquaculture Society 30, 90–97.
Arkush K.D., Cherr G.N. & Clegg J.S. (2008) Induced thermotolerance and tissue Hsc70 in juvenile Coho salmon, Oncorhynchus kisutch. Acta Zoologica 89, 331–338.
Balucci C.A. (2005) Studies on the properties of an extract of
Opuntia ficus indica. Ph.D thesis, University of Malta, 376 pp.
Benli A.Ç.K., Köksal G. & Özkul A. (2008) Sublethal ammonia
exposure of Nile tilapia (Oreochromis niloticus L.): effects on
gill, liver and kidney histology. Chemosphere 72, 1355–1358.
Bhakta J.N. (2006) Ammonia toxicity to four freshwater fish
species: Catla catla, Labeo bata, Cyprinus carpio and Oreochromis mossambica. Electronic Journal of Biology 2, 39–41.
Brown M.A., Upender R.P., Hightower L.E. & Renfro J.L.
(1992) Thermoprotection of a functional epithelium: heat
stress effects on transepithelial transport by flounder renal
tubule in primary monolayer culture. Proceedings of the National Academy of Sciences USA 89, 3246–8250.
Brown H.M., Briden A., Stokell T., Griffin F.J. & Cherr G.N.
(2004) Thermotolerance and Hsp70 profiles in adult and
embryonic California native oysters, Ostreola conchaphila
(Carpenter, 1857). Journal of Shellfish Research 23,
135–141.
Camilleri T. (2002) The prophylactic effect of TEX-OE in
angelfish (Pterophyllum scalare) against stress caused by
ammonia, nitrite and chlorine. B.Sc Hons thesis, University of
Malta, 124 pp.
Clegg J.S., Uhlinger K.R., Jackson S.A., Cherr G.N., Rifkin E. &
Friedman C.S. (1998) Induced thermotolerance and the heat
shock protein-70 family in the Pacific oyster Crassostrea gigas.
Molecular Marine Biology and Biotechnology 7, 21–30.
Clegg J.S., Jackson S.A., Hoa N.V. & Sorgeloos P. (2000)
Thermal resistance, developmental rate and heat shock proteins in Artemia franciscana, from San Francisco Bay and
Southern Vietnam. Journal of Experimental Marine Biology and
Ecology 252, 85–96.
de la Vega E., Hall M.R., Degnan B.M. & Wilson K.J. (2006)
Short-term hyperthermic treatment of Penaeus monodon increases expression of heat shock protein 70 (HSP70) and
Journal of Fish Diseases 2012, 35, 563–568
reduces replication of gill associated virus (GAV). Aquaculture
253, 1–4.
Duncan R.F. (2005) Inhibition of Hsp90 function delays and
impairs recovery from heat shock. FEBS Journal 272, 5244–
5256.
Sandilands J., Drynan K.D. & Roberts R.J. (2009) Preliminary
studies on the enhancement of storage time of chilled milt of
Atlantic salmon, Salmo salar L., using an extender containing
the TEX-OE heat shock-stimulating factor. Aquaculture
Research 40, 402–409.
Feder M.E. & Hoffman G.E. (1999) Heat shock proteins,
molecular chaperones, and the stress response: evolutionary
and ecological physiology. Annual Review of Physiology 61,
243–282.
Santhi K., Gunasekhar K., Babu C.S., Reddy K.P.O. & Neeraja
P. (1992) Changes in the dehydrogenase levels of fish Oreochromis mossambicus under chronic ammonia stress. Journal of
Inland Fisheries Society of India 24, 71–73.
Hartl F.U. & Hayer-Hartl M. (2009) Converging concepts of
protein folding in vitro and in vivo. Nature Structural and
Molecular Biology 16, 574–581.
Schuler D.J., Boardman G.D., Kuhn D.D. & Flick G.J. (2010)
Acute toxicity of ammonia and nitrite to Pacific white shrimp,
Litopenaeus vannamei, at low salinities. Journal of the World
Aquaculture Society 41, 438–441.
Israeli-Weinstein D. & Kimmel E. (1998) Behavioral response of
carp (Cyprinus carpio) to ammonia stress. Aquaculture 165,
81–93.
Laemmli U.K. (1970) Cleavage of structural proteins during
assembly of the head of bacteriophage T4. Nature, 227, 680–
685.
Mchaourab H.S., Godar J.A. & Stewart P.L. (2009) Structure
and mechanism of protein stability sensors: chaperone activity
of small heat shock proteins. Biochemistry 48, 3828–3837.
Pockley A.G. (2003) Heat shock proteins as regulators of the
immune response. Lancet 362, 469–476.
Pockley A.G., Muthana M. & Calderwood S.K. (2008) The dual
immunoregulatory roles of stress proteins. Trends in Biochemical Sciences 33, 71–79.
Robert J. (2003) Evolution of heat shock protein and immunity.
Developmental and Comparative Immunology 27, 449–464.
Roberts R.J., Agius C., Saliba C., Bossier P. & Sung Y.Y. (2010)
Heat shock proteins (chaperones) in fish and shellfish and their
potential role in relation to fish health: a review. Journal of Fish
Diseases 33, 789–801.
Russo R.C. 1985. Ammonia, nitrite, and nitrate. In: Fundamentals of Aquatic Toxicology (ed. by G.M. Rand & S.R.
Petrocelli), pp. 455–474. Hemisphere Publishing, Washington
DC.
Ryckaert J., Pasmans F., Tobback E., Duchateau L., Decostere
A., Haesebrouck F., Sorgeloos P. & Bossier P. (2010) Heat
shock proteins protect platyfish (Xiphophorus maculatus) from
Yersinia ruckeri induced mortality. Fish and Shellfish Immunology 28, 228–231.
2012
Blackwell Publishing Ltd
Y Y Sung et al. Hsp70 and ammonia toxicity
568
Sung Y.Y., Van Damme E.J.M., Sorgeloos P. & Bossier P.
(2007) Non-lethal heat shock protects gnotobiotic Artemia
franciscana larvae against virulent vibrios. Fish and Shellfish
Immunology 22, 318–326.
Sung Y.Y., Pineda C., MacRae T.H., Sorgeloos P. & Bossier P.
(2008) Exposure of gnotobiotic Artemia franciscana larvae to
abiotic stress promotes heat shock protein 70 synthesis and
enhances resistance to pathogenic Vibrio campbellii. Cell Stress
and Chaperones 13, 59–66.
Sung Y.Y., Ashame M.F., Chen S.J., MacRae T.H., Sorgeloos P.
& Bossier P. (2009a) Feeding Artemia franciscana (Kellogg)
larvae with bacterial heat shock protein, protects from Vibrio
campbellii (Baumann) infection. Journal of Fish Diseases 32,
675–685.
Sung Y.Y., Dhaene T., Defoirdt T., Boon N., MacRae T.H.,
Sorgeloos P. & Bossier P. (2009b) Ingestion of bacteria overproducing DnaK attenuates Vibrio infection of gnotobiotic Artemia franciscana larvae. Cell Stress and Chaperones 14, 603–609.
Todgham A.E., Schulte P.M. & Iwama G.K. (2005) Cross-tolerance in the tidepool sculpin: the role of heat shock proteins.
Physiological and Biochemical Zoology 78, 133–144.
Wiese J., McPherson S., Odden M.C. & Shlipak M.G. (2004)
Effect of Opuntia ficus indica on symptoms of the alcohol
hangover. Archive of Internal Medicine 164, 1334–1340.
Received: 4 January 2011
Revision received: 11 March 2011
Accepted: 7 July 2011