Journal of Invertebrate Pathology 111 (2012) 20–26

Contents lists available at SciVerse ScienceDirect

Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Physiological performance of juvenile Haliotis rufescens and Haliotis discus

hannai abalone exposed to the withering syndrome agent
Roxana C. González a,b, Katherina Brokordt b,a,⇑, Karin B. Lohrmann a
a
Universidad Católica del Norte, Facultad de Ciencias del Mar, Larrondo 1281, Coquimbo, Chile
b
Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Larrondo 1281, Coquimbo, Chile

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

Article history: Withering syndrome (WS) is a serious chronic disease caused by infection with the bacterium Candidatus
Received 19 January 2012 Xenohaliotis californiensis, a Rickettsiales-like organism (WS-RLO) that affects multiple abalone species
Accepted 14 May 2012 in both natural and farmed populations. However, there is no available information regarding the effects
Available online 23 May 2012
of this disease on the physiological performance of infected abalone. We studied the effect of different
levels of infection on components of energy balance and physiological indices (rates of absorption and
Keywords: assimilation, O/N ratio, and scope for growth) in the abalone species Haliotis rufescens and Haliotis discus
Withering syndrome
hannai. Juveniles were exposed to C. X. californiensis transmission for 130 days, during which time the
Candidatus Xenohaliotis californiensis
Abalone
presence/absence of WS-RLOs was evaluated by PCR (following DNA sequencing-based confirmation of
Haliotis rufescens 100% identity with the sequence of C. X. californiensis from California), and the prevalence and intensity
Haliotis discus hannai of infection were evaluated via histological analysis. Among H. rufescens juveniles exposed to the bacte-
Physiological performance rium, 92% became infected (positive by histology), and the intensity of infection ranged from low (degree
1) to moderate (degree 2). In contrast, no H. discus hannai juveniles were positive for WS-RLO by histol-
ogy, although 23% were positive by PCR, possibly indicating incipient WS-RLO infection that did not
develop during the experimental period or to mere presence of WS-RLO DNA in the sample. Infection
of H. rufescens juveniles by WS-RLOs negatively affected all components of the energy balance and phys-
iological indices, such as scope for growth and the O/N ratio, in direct relation to the degree of infection.
The most strongly affected functions were the rate of ingestion, standard metabolism, and production of
feces, which were reduced by 60–80% in the most highly infected individuals. The reduced energy intake
in the organisms produced a strong energy imbalance such that the energy available for growth was
reduced by 49% in infected organisms. In contrast, juveniles of H. discus hannai carrying the bacterium
developed no infection and showed no alterations of physiological function. Our results indicate that
the level of early infection by WS-RLOs may exert a negative effect on physiological activity in H. rufes-
cens, even when the disease is not evident.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction californiensis, a Rickettsiales-like organism (WS-RLO). The trans-

Withering syndrome (WS) is a serious chronic disease that af-
fects various abalone (Haliotis spp.) species in both natural and
farmed populations. The disease was first observed in the 1980s
in the Channel Islands southwest of California, where it caused a
99% decline in the population of the black abalone H. cracherodii.
Since that time, this disease has been detected in pink (H. corrugat-
a), flat (H. walallensis), white (H. sorenseni), red (H. rufescens), green
(H. fulgens), and Taiwanese (H. diversicolor supertexta) abalones
(Friedman et al., 2002; Wetchateng et al., 2010). The etiological
agent of WS is the intracellular bacterium Candidatus Xenohaliotis
⇑ Corresponding author at: Centro de Estudios Avanzados en Zonas Áridas
(CEAZA), Larrondo 1281, Coquimbo, Chile.
E-mail addresses: rgo006@alumnos.ucn.cl (R.C. González), kbrokord@ucn.cl (K.
Brokordt), klohrman@ucn.cl (K.B. Lohrmann).
mission of WS is direct and horizontal via the oral–fecal route
(Moore et al., 2001a). Susceptibility to WS and its expression differ
among abalone species and are influenced by the ambient temper-
ature among other factors (Moore et al., 2009). The clinical signs of
WS are characterized by morphological changes in the digestive
gland, such as metaplasia (cells involved in secretion/absorption
are replaced by transport ducts), and/or degeneration of gastroin-
testinal tissue in the form of atrophy of tubules, increases in con-
nective tissue, and inflammation. These morphological changes
lead to a loss of functionality of the digestive system, resulting in
the animal entering a state of forced inanition that obliges it to
catabolize its energy reserves and eventually the structural pro-
teins of its pedal muscle, which are replaced by connective tissue
(Gardner et al., 1995; Friedman et al., 1997; Braid et al., 2005).
Thus, the changes that occur in these tissues during development
of the disease are similar to those associated with starvation:

0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jip.2012.05.005
 R.C. González et al. / Journal of Invertebrate Pathology 111 (2012) 20–26
weakness, lethargy, constriction of body mass, and finally death
(Moore et al., 2001a). Despite the effects of the disease on the func-
tion of the digestive tissues, little information is available regard-
ing the associated effects on the physiological activity of
abalones, specifically with respect to the efficiency of the use of
available energy (Kismohandaka et al., 1993; Rosenblum et al.,
2005).
The activity of an organism during essential processes, such as
growth and reproduction, is determined by the balance among
the processes involved in incorporation, and the use and loss of
available energy, i.e., the energy balance. From the components
of the energy balance, we can estimate several indices of efficiency
and physiological condition as indicators of the state of an organ-
ism at a given moment. These indicators are highly sensitive and
allow anticipation of effects on growth, reproduction, and survival.
Several studies have reported that the presence of parasites and/or
pathogens in marine mollusks alters their energy balance, leading
to a diminution or loss of the animal’s capacity for growth or repro-
duction (Barber et al., 1988; Newell, 1985; Kennedy et al., 1995;
Flye-Sainte-Marie et al., 2007). While the marked changes pro-
voked by WS may indicate that associated physiological alterations
occur in infected organisms, the dynamics of this response during
the evolution of infection are not currently understood, especially
during its initial phases in which clinical signs are not evident.
Obtaining an understanding of this process is fundamental, partic-
ularly because infection by WS-RLOs may persist in a latent state
for long periods of time (e.g., at low temperatures) without devel-
opment of disease.
The abalone farming industry has undergone robust worldwide
growth in recent years, mainly due to its high commercial value
and the decline of natural stocks due to overexploitation (Gordon
and Cook, 2001). In Chile, abalone production has increased mark-
edly in the last decade (Flores et al., 2007), though because this re-
source does not exist in the natural environment, in this region, the
industry is based on the cultivation of only two species: the red
abalone, H. rufescens, and the Pacific abalone, H. discus hannai.
WS has become a threat to abalone farming in Chile and world-
wide. Among abalone species, the red abalone is the third most
susceptible to the disease; information regarding the susceptibility
of the Pacific abalone to WS is not available. Because the conditions
employed in cultivation are extremely favorable for the develop-
ment of infectious diseases, it is important to determine the effects
of this pathogen on the physiological performance of these animals
and therefore on production traits, such as growth and reproduc-
tive capacity. The objective of the present study was to evaluate
the effects of different degrees of infection by C. X. californiensis
on the physiological activity of juveniles of the abalone species
H. rufescens and H. discus hannai.
2. Materials and methods
2.1. Collection and maintenance of animals
Juveniles of the abalone species H. rufescens and H. discus hannai
(N = 300/species) with a shell length of between 15.0 and 35.7 mm
(22.5 mm mean shell length) and between 15.5 and 33.9 (22.6 mm
mean shell length), respectively; and free of WS-RLOs were ob-
tained from the Center for Abalone Cultivation of the Universidad
Católica del Norte, Coquimbo, Chile. Additionally 150 H. rufescens
adults (shell length 8 cm), with WS (as indicated by an atrophied
foot muscle and histological diagnosis, which showed moderate
and severe infection intensity levels), were donated by an abalone
farm from the Coquimbo Region. The juveniles were divided into
six groups (three treatment and three control groups, N = 45/
group), which were distributed among 12 10-L aquaria provided
21
with aeration and a constant seawater flow (microfiltrated at
0.45 lm) at a salinity of 34.6 psu, and fed ad libitum with the
macro-alga Macrocystis pyrifera. All individuals were acclimated
for 14 d at 18 °C under the aquarium conditions. Following accli-
mation, the initial histological and physiological condition of the
abalone was evaluated (N = 15 individuals per species).
2.2. Exposure to WS-RLP and sampling
The juveniles of both species in the treatment groups were ex-
posed to horizontal transmission of WS-RLO via continuous expo-
sure to the effluent from the head pond, which contained 50 red
abalone infected with WS-RLOs. The juveniles in the control groups
were supplied directly with seawater microfiltered through a
0.45 lm filter (Fig. 1). For the purpose of obtaining individuals with
several levels of infection, we collected and analyzed samples
(N = 5 individuals/species/treatment or control group) five times
during the experimental period, at 10, 20, 30, 94, and 130 d follow-
ing the initiation of exposure to WS-RLOs. For each sample, the
presence of C. X. californiensis was examined by means of (1) his-
tological observation of tissue; and (2) PCR analysis to exclude the
presence of histologically undetectable bacteria. We further evalu-
ated the effects of infection by C. X. californiensis on the compo-
nents of energy balance and performance of the physiological
indices.
2.3. Measurement of components of energy balance and estimation of
physiological indices
The components of the energy balance were determined using
the equation C = P + F + R + U (Ricker, 1968), where C represents
the energy consumed as food, P the scope for growth or the energy
available for growth, F the energy lost via feces, R the energy used
for oxygen consumption, and U the energy lost via excretion of
ammonia. Each component of the energy balance was measured
individually, according to the methods described by González
et al. (2010), and was transformed into an energy equivalent. Food
consumption (C) was estimated as the difference between the
mass of algae supplied as food and that remaining after a 72-h per-
iod. A control tank containing no animals was used to evaluate
changes in algal mass due to erosion or hydration. Fecal production
(F) was measured over a 12-h period; feces were filtered and
washed with 4% ammonium formate to eliminate salts. Both the
remaining food and feces produced were dried to constant mass
(48 h at 70 °C). The food consumption and fecal production values
were transformed into energy equivalents using the following con-
version factors: 1 g feces = 2817 cal (Peck et al., 1987), and 1 g of M.
pyrifera = 3225 cal (Lamares and Wing, 2001). The respiratory rate
(R) was determined as the difference between the initial and final
oxygen levels measured over a 1.5-h period of incubation of an ani-
mal in a sealed respiratory chamber at oxygen saturation. The oxy-
gen concentration in the samples was estimated using the method
of Winkler (1888). The oxygen consumption values were trans-
formed into energy equivalents using the conversion factor of
Thompson and Bayne (1974), where 1 mL oxygen = 4.75 cal. The
rate of ammonia excretion (U) was measured as the difference be-
tween the initial and final ammonia concentrations in samples col-
lected parallel to those for oxygen determination (i.e., incubation
for 1.5 h). The ammonia concentrations in the samples were esti-
mated via the phenol–hypochlorite method of Solorzano (1969).
A control (a chamber with no abalone under the same conditions)
was used to evaluate the normal fluctuations of both oxygen and
dissolved ammonia in the aqueous incubation medium. The
ammonia excretion values were transformed into energy equiva-
lents using the conversion factor of Elliott and Davidson (1975)
in which 1 mg NH4-N = 5.94 cal.
22 R.C. González et al. / Journal of Invertebrate Pathology 111 (2012) 20–26
Exposed to WS-RLP
n=50 n=45
infected n=45
H.rufescens
adults
Unexposed to WS-RLP
n=45
n=45
Haliotis rufescens
Haliotis discus hannai
Fig. 1. Tanks and aquaria distribution within the experimental unit. Haliotis rufescens (dotted pattern) and Haliotis discus hannai (uniform pattern) juvenile abalone exposed
and unexposed to Candidatus Xenohaliotis californiensis (WS-RLO). Seawater supply for exposed juveniles passes through the black tank containing the infected H. rufescens
adults. Seawater supply for unexposed juveniles passes through the white tank with no abalone. Dotted arrows indicate the water flux direction.
The efficiencies of the absorption (EAb) and assimilation (EAs)
of food were estimated based on the energy equivalents described
in the equations presented by Ricker (1968), in which
EAb = [(C F)/C]  100, and EAs = [(C (F + U))/C]  100.
Scope for growth was calculated based on the energy balance
equation as P (or SFG) = C (F + R + U).
The O/N ratio (a measure of the balance of catabolic processes
and an indicator of the stress state) was calculated based on the
atomic equivalents of oxygen consumed and nitrogen excreted.
2.4. Histological analysis
Sampled tissues (gills, digestive gland, post-esophagus, and
nephridia) were fixed in Davidson’s for 24 h (Shaw and Battle,
1957), dehydrated and cleared in a tissue processor, embedded in
paraplast, sectioned to a thickness of 5 lm, and stained with hema-
toxylin–eosin for observation under a light microscope. Quantifica-
tion of the WS-RLO load in the digestive tract was performed based
on the scale of Friedman et al. (1997), which assigns three degrees
of infection intensity according to the number of bacterial inclu-
sions in a 200 field of magnification: degree 0 = absence, degree
1 = 1–10, degree 2 = 11–100, and degree 3 = >100 inclusions per
field.
2.5. PCR and sequence analyses
Samples were obtained from the post-esophagus and processed
according to the extraction method described in the AxyPrep
Multisource Genomic DNA Miniprep Kit. DNA amplification was
performed using the RA 5-1 and RA 3-6 primers for C. X.
n=45 n=45
n=45 n=45
n=45 n=45
n=45 n=45
californiensis DNA, which amplify a 160-bp fragment of the small
subunit of the bacterial ribosome. PCR amplification was per-
formed in a standard reaction volume of 50 lL containing 1X PCR
buffer (Invitrogen), 1.5 mM magnesium chloride (MgCl2), 0.2 mM
nucleotide triphosphates (dNTPs), 0.5 lM each primer, 1.6 U of
Taq polymerase, and 5 lL of DNA template (Andree et al., 2000).
The amplification conditions were as follows: initial denaturation
at 95 °C for 5 min, followed by 40 cycles of 95 °C for 1 min, 62 °C
for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min.
The obtained DNA was resolved using vertical electrophoresis in
a polyacrylamide gel (12% acrylamide, 0.32% bisacrylamide) and
stained with silver nitrate. For confirmation that the PCR product
corresponded to the 16S rDNA fragment of C. X. californiensis, rep-
resentative samples were sent to the Molecular Diversity Unit of
Pontificia Universidad Católica de Chile for nucleotide sequence
analysis. The obtained sequences were compared with the known
sequence of the 16S rDNA gene (GenBank nucleotide sequence ID
AF133090) via multiple sequence alignment using ClustalW2
(Thompson et al., 1994) to confirm 100% identity with the WS-
RLO sequence from California.
2.6. Statistical analyses
To evaluate the effect of infection by WS-RLO on the physiolog-
ical activity of H. rufescens juveniles, individuals were grouped
according to the degree of infection, independent of the time at
which they had been sampled. The relationship between the de-
gree of WS-RLO infection and each component of the energy bal-
ance and the physiological indices was evaluated via Pearson’s
linear correlation after confirmation of normality of the data and
 R.C. González et al. / Journal of Invertebrate Pathology 111 (2012) 20–26
homoscedasticity of variance (using Kolmogorov–Smirnov and
Cochrane tests, respectively).
For H. discus hannai, since only PCR-positive individuals were
identified, and no histologically positive individuals were found,
the existence of significant differences between PCR-positive and
PCR-negative individuals was evaluated by one-way analysis of
variance (ANOVA).
3. Results
3.1. Prevalence and intensity of infection of WS-RLOs
Basophilic inclusions (violet or purple) with spherical to oval
forms were observed in cells of the gastrointestinal epithelium of
H. rufescens. The bacterial inclusions were usually situated apical
to the nucleus of the host cell (Fig. 2). Among the red abalone indi-
viduals exposed to the bacterium during the experiment, 92% were
infected, i.e., histology-positive, and also were PCR-positive.
Histology-positive animals showed infection intensities ranging
from low (degree 1) to moderate (degree 2). In H. discus hannai,
however, no histologically positive individuals were found,
although 23% were PCR positive.
3.2. Effect of infection by WS-RLO on components of energy balance
and physiological indices
In H. rufescens juveniles, the rate of ingestion (C), the production
of feces (F), and the respiration rate (R) showed a strong negative
correlation (70–80%) with the degree of WS-RLO infection
(Fig. 3). The most highly infected individuals showed reductions
of approximately 60–80% in these physiological rates. In compari-
son, correlation of the ammonia excretion rate (U) with the degree
of infection, although significant, was weaker (Fig. 3). Conversely,
the efficiencies of both absorption (EAb) and assimilation (EAs) in-
creased significantly with increases in the degree of infection of the
abalone, and showed a significantly correlation of 30% (Fig. 4).
Despite these greater efficiencies, the scope for growth (SFG) de-
creased significantly with an increasing degree of infection, and
the O/N ratio was negatively correlated with the degree of infec-
tion by WS-RLOs in a similar fashion (Fig. 4).
Because juveniles of H. discus hannai were found to be positive
for WS-RLOs only by PCR, analysis of the effect of WS-RLOs
Fig. 2. Candidatus Xenohaliotis californiensis (WS-RLO) inclusion (arrowhead) in
postesophagus of red abalone Haliotis rufescens, in a supranuclear location in the
epithelial host cell. Note the very finely granular and homogenous texture of the
inclusion. Stain: H & E.
23
presence on the components of the energy balance and the physi-
ological indices was performed by grouping PCR-positive and PCR-
negative individuals. However, no significant (p < 0.05) differences
were found via ANOVA between the PCR-positive and PCR-
negative groups for any of the components of the energy balance
or physiological indices (Table 1).
4. Discussion
Infection by C. X. californiensis negatively affected the physio-
logical activity of juveniles of H. rufescens in direct correlation to
the degree of infection. The most strongly affected functions were
the rates of feeding, standard metabolism, and production of feces.
The reduction of energy uptake by the organisms brought about a
strong alteration of the energy balance such that the energy avail-
able for growth was reduced by up to 49% in the most strongly af-
fected individuals. In contrast, although 23% of juveniles of H.
discus hannai carried the bacterium DNA, no individuals of this spe-
cies developed an infection, and correspondingly, no alterations of
their physiological functions were detected.
The use of energy may vary according to the conditions con-
fronted by an organism at a given moment. Parasitism by C. X. cal-
iforniensis could cause alterations in the physiological functions of
the red abalone via two routes: (1) by directly causing alterations
in digestive tissues, thereby affecting digestive functions and, con-
sequently, decreasing the incorporation of energy for the functions
of growth, reproduction, and maintenance, which is observed in all
cases of advanced disease as a consequence of metaplasia of diges-
tive gland tissue; and (2) via generating a state of general stress
that alters physiological or behavioral functions (e.g. feeding).
Because the bacteria reside within cells of the digestive tract it
is likely that the functions primarily affected will be associated
with digestive capacities. In this study, the incorporation of energy
from food was reduced by 62% in H. rufescens individuals showing
the highest degree of infection by WS-RLO detected in this study.
However, no negative correlation was found between the efficien-
cies of food absorption and assimilation (reflecting digestive func-
tion) and the degree of infection. On the contrary, infected abalone
showed 35–37% increase in absorption and assimilation efficien-
cies, respectively, suggesting that the low food intake may have
been compensated for via increasing both efficiencies in unaffected
digestive tissues. This type of physiological compensation has been
observed in other gastropods, such as Chorus giganteus, which
exhibits increased absorption efficiency when its rate of food in-
take decreases (Navarro et al., 2002). Besides, with a diminution
of the ingestion rate in juveniles of H. rufescens, the production of
feces and excretion of ammonia also diminished at higher levels
of infection by WS-RLOs, which would be explained by increased
efficiencies of absorption and assimilation, respectively. In addi-
tion, an appreciable decrease in the O/N ratio was observed with
an increasing degree of infection. This index indirectly indicates
the state of stress of individuals, with a lower value indicating a
higher level of stress and the use of mainly proteins in preference
to lipids and carbohydrates as energy substrates compared to unaf-
fected individuals (Mayzaud and Conover, 1988). Thus, the reduc-
tion in the rate of food intake could have been a consequence of a
state of stress induced by parasitism, rather than a diminution of
the digestive capacity.
Our results indicate that infection by WS-RLOs provoked a
general reduction of metabolism in juveniles of H. rufescens.
The consumption of oxygen and the excretion of ammonia are
indicators of the basal metabolism of an organism; the former
decreased by up to 61% in infected abalone and the second by
up to 45%. Decreased consumption of oxygen has been described
as a compensatory mechanism in some mytilids for conserving
24 R.C. González et al. / Journal of Invertebrate Pathology 111 (2012) 20–26

Food intake Fecal production

y = -7.4 x + 31 25 y = - 4.1 x + 14
40 r = -0.7 r = -0.8
p = 0.000 p = 0.000
35 20
30
Cal.g-1h-1

Cal.g-1h-1
15
25
20
10
15
10 5
5
0

Oxygen consumption Ammonia excretion

2.5 0.9
y = -0.4 x + 1.7 y = -0.1 x + 0.5
r = -0.8 0.8 r = -0.4
p = 0.000 p = 0.003
2.0 0.7
0.6
Cal.g-1h-1

Cal.g-1h-1
1.5
0.5
0.4
1.0
0.3

0.5 0.2
0.1
0.0 0
0 1 2 0 1 2
WS-RLP infection intensity WS-RLP infection intensity

Fig. 3. Effects of different infection intensities by Candidatus Xenohaliotis californiensis on physiological rates associated to the energy balance of Haliotis rufescens juveniles.
n = 37, 8 and 20, for 0, 1 and 2 WS-RLO intensity of infection.

Scope for growth O/N Ratio

30 y = -2.8 x + 14 30 y = -1.5 x + 8.4

r = -0.3 r = -0.4
25 p = 0.005 25 p= 0.013

20 20
Cal.g-1h-1
Cal.g-1h-1

15 15

10 10

5 5

0 0

5
10

Absorption efficiency Assimilation efficiency

y = 8.7 x + 40 y = 8.4 x + 39
100 r = 0.3 100 r = 0. 3
p = 0.016 80 p = 0.029
80
60 60
40 40
20
(%)

20
(%)

0 0
-20 -20
-40 -40
-60 -60
-80 0 1 2 -80 0 1 2
WS-RLP infection intensity WS-RLP infection intensity

Fig. 4. Effects of different infection intensities by Candidatus Xenohaliotis californiensis on physiological indices and efficiencies of Haliotis rufescens juveniles. n = 37, 8 and
20, for 0, 1 and 2 WS-RLO intensity of infection.

energy in situations where ingestion rates are decreased
(Navarro et al., 2002). Moreover, as a direct result of a sharp
reduction in the incorporation of food, it is reasonable to expect
a decrease in oxygen consumption (which reflects the oxidation
of carbon compounds assimilated by an animal), inducing this
metabolic depression. However, in the context of the energy bal-
ance, a decreased loss of energy via metabolism may reflect an
enhancement of the efficiency of energy usage (Navarro et al.,
2002) due to a decreasing expenditure of energy for carrying
out metabolic work, thereby making more energy available for
somatic growth and reproduction (in the case of adult organ-
isms). However, this effect may also be an indicator of physio-
logical deterioration in an animal (Flye-Sainte-Marie et al.,
2007). Our results are in better accord with this second option;
upon reducing their rate of food consumption, infected organ-
isms also reduce the rate at which they transform the food en-
ergy, as reflected in a slower metabolism, without increasing
their metabolic efficiency.
 R.C. González et al. / Journal of Invertebrate Pathology 111 (2012) 20–26
Table 1
Physiological rates, indices and efficiencies (mean ± SE) of Haliotis discus hannai
juveniles positive and negative for WS-RLP by PCR. n = 15 and 50, for PCR positive and
negative juveniles, respectively.
Physiological parameters PCR positive (SE) PCR negative (SE)
Food intake (Cal/g/h) 17 (2.7) 13 (1.0)
Oxygen consumption (Cal/g/h) 1.2 (0.1) 1.1 (0.1)
Ammonia excretion (Cal/g/h) 0.3 (0.1) 0.2 (0.0)
Fecal production (Cal/g/h) 7.4 (1.3) 6.2 (0.9)
SFG (Cal/g/h) 7.9 (0.1) 4.7 (0.9)
O/N ratio 8.6 (2.8) 10 (2.0)
Absorption efficiency (%) 63 (10) 56 (4.9)
Assimilation efficiency (%) 65 (4.8) 51 (5.1)
As a consequence of the sharp decreases in food intake and
metabolism in infected H. rufescens individuals, their potential for
growth likewise declined significantly in direct correlation with
the level of infection. The rate of food intake (entry of energy into
the organism) is one of the most important components of the en-
ergy balance, and in this case, its reduction led to a reduction in the
proportion of energy available for growth in juveniles by 49% in the
most infected individuals, even though the energy losses via
metabolism were lower in the latter group. Several studies in
marine mollusks have found that infectious diseases can alter the
energy balance of the host (Newell, 1985; Barber et al., 1988;
Kismohandaka et al., 1993; Kennedy et al., 1995; Villalba et al.,
2004; Rosenblum et al., 2005; Flye-Sainte-Marie et al., 2007). The
magnitude of these changes and their effects depend on the viru-
lence of the parasite, the extent of disease development, and the
resistance of the host. In the case of WS, there are no previous re-
ports addressing these types of alterations. Our results indicate
that there is a negative relationship between the physiological
functions of H. rufescens and the WS-RLOs infection level. The func-
tions that were most affected were the rates of ingestion and
metabolism. Loss of feeding capacity has been previously observed
only in individuals manifesting signs of disease over long time
periods (Moore et al., 2001b). In the present study, we observed
slight signs of WS, a low degree (0–10%) of metaplasia in the diges-
tive gland, minor body shrinkage, and low to moderate infection
intensities. These observations indicate that in juveniles, the signs
of WS need not have developed to the maximum degree to provoke
stress and physiological deterioration in an animal and that the
physiological rates we evaluated represent good early indicators
of the presence of the disease.
In this study, among individuals exposed to C. X. californiensis
(WS-RLO), 92% of H. rufescens juveniles became infected (i.e. his-
tology-positive) during the 130-d experimental period. In con-
trast, the bacterium was detected in only 23% of H. discus
hannai juveniles (PCR-positive), and this species did not develop
bacterial inclusions (histology-negative), .possibly indicating
incipient infections by C. X. californiensis that did not fully de-
velop during the experimental period or to mere presence of
WS-RLO DNA in the sample. These results are in accord with
the absence of alterations in physiological functions observed
in the juveniles of this species. Therefore, our results indicate
that there are sharp differences between the two abalone species
regarding their susceptibility to developing infection by C. X.
californiensis.
Various authors have indicated that WS is modulated by tem-
perature and that this property is a key factor in development of
the disease in animals infected with WS-RLOs (Friedman et al.,
1997; Moore et al., 2000). Studies have shown that some species
of abalone may remain infected with the bacterium for long peri-
ods without exhibiting disease development and that the temper-
ature at which WS is induced varies widely among species
(Friedman et al., 1997; Moore et al., 2000; Braid et al., 2005; Vilchis
25
et al., 2005; García-Esquivel et al., 2007). For example, H. rufescens
individuals exposed to WS-RLOs at 18 °C for 200 d expressed clin-
ical signs of WS associated with 33% mortality, whereas individuals
maintained at 14 °C did not, with the latter group presenting zero
mortality and only minor signs of WS (Moore et al., 2000). In a
long-term experiment (730 d), Vilchis et al. (2005) compared the
effects of the quality and quantity of food and temperature on
the development of WS in H. rufescens and H. fulgens and concluded
that the former species was highly susceptible to WS at 18 °C,
whereas H. fulgens was not. Moreover, their results indicated that
H. fulgens might be more resistant to the pathogen under condi-
tions similar to an El Niño event in California. Another study
(García-Esquivel et al., 2007) indicated that H. fulgens maintained
at 25 °C do display the signs of WS, whereas temperatures
P25 °C could cause thermal stress, thereby contributing to physi-
ological deterioration of the organism and therefore vulnerability
to WS-RLOs. Contrary to the hypothesis of ‘‘thermal stress to the
host’’ as a factor triggering WS, Moore et al. (2009) proposed that
the temperature at which clinical signs of WS are expressed is
more closely related to the preferred temperature of each species.
The preferred temperature of ectotherms, i.e., that chosen by ani-
mals in a temperature gradient, reflects the optimal temperature
for many biological processes, such as metabolism, movement,
reproduction, and growth (Jobling, 1981). In this context, it has
been found that the temperature preferred by H. rufescens is
18.8 °C, while the optimal temperature for the growth of juveniles
of this species (25 mm) is only 18.4 °C (Díaz et al., 2000). Coinci-
dentally, Díaz et al. (2006) reported that the preferred temperature
of H. fulgens is 25.4 °C. Thus, the preferred temperature for both
species is the same as that at which the signs of WS develop. It is
likely that the temperature preferred by H. discus hannai is higher
than that used in our experiment (18 °C), as the optimum growth
temperature reported for this abalone species is 20 °C (Xusheng
et al., 1990). This species may therefore be vulnerable to WS at a
temperature higher than that used in the present study.
In general, the differences observed among different abalone
species in the expression of WS make it clear that elevated tem-
perature alone may not hasten the development of WS-RLOs
simply by accelerating the rate of replication of the bacterium
or by stressing the host, i.e. increasing its susceptibility to WS-
RLOs. The specific role of temperature in the expression of WS
and other factors that may favor the development of C. X. califor-
niensis remain to be investigated among abalone species (Moore
et al., 2009). In the specific case of H. discus hannai, future stud-
ies should investigate the conditions (e.g., higher temperatures)
under which this species may be susceptible to the development
of WS.
The present study reveals that unconventional methods, such as
employing physiological indicators, may be useful for detecting
preclinical signs of WS on abalone farms. In addition to their low
cost and ready implementation, these indicators deliver a rela-
tively complete view of the state of an organism without the neces-
sity of sacrificing the animal in the process. Among these
indicators, decreases in the rate of food intake are the most easily
evaluated clinical disease signs.
Acknowledgments
We are grateful to the Centro de Producción de Abalón de la
Universidad Católica del Norte (CPA-UCN) for their invaluable con-
tribution in providing all the necessary facilities required to under-
take this study under controlled conditions. We also thank Andrea
Hueche and Ana Valdivia for their valuable support during labora-
tory work. This study was supported by FONDEF Grant D02I1129
and Thesis Fund of Coquimbo Regional Government.
26 R.C. González et al. / Journal of Invertebrate Pathology 111 (2012) 20–26

References Mayzaud, P., Conover, R., 1988. O:N atomic ratio as a tool to describe zooplankton
metabolism. Mar. Ecol. Prog. Ser. 45, 289–302.
Moore, J., Robbins, T., Friedman, C., 2000. Withering syndrome in farmed red
Andree, K., Friedman, C., Moore, J., Hedrick, R., 2000. A polymerase chain reaction for
abalone Haliotis Rufescens: thermal induction and association with a
detection of genomic DNA of a Rickettsiales-like prokaryote associated with
gastrointestinal Rickettsiales-like prokaryote. J. Aquat. Anim. Health 12, 26–34.
withering syndrome in black abalone (Haliotis cracherodii). J. Shellfish Res. 19,
Moore, J., Cherr, G., Friedman, C., 2001a. Detection of Candidatus Xenohaliotis
213–218.
californiensis (Rickettsiales-like prokaryote) inclusions in tissue squashes of
Barber, B., Ford, S., Haskin, H., 1988. Effects of the parasite MSX (Haplosporidium
abalone (Haliotis spp.) gastrointestinal epithelium using a nucleic acid
nelsoni) on oyster (Crassostrea virginica) energy metabolism. I. Condition index
fluorochrome. Dis. Aquat. Org. 46, 147–152.
and relative fecundity. J. Shellfish Res. 7, 25–31.
Moore, J., Robbins, T., Hedrick, R., Friedman, C., 2001b. Transmission of the
Braid, B., Moore, J., Robbins, T., Hedrick, R., Tjeerdema, R., Friedman, C., 2005. Health
rickettsiales-like prokaryote ‘‘Candidatus Xenohaliotis californiensis’’ and its
and survival of red abalone, Haliotis rufescens, under varying temperature, food
role in withering syndrome of California abalone, Haliotis Spp.. J. Shellfish Res.
supply, and exposure to the agent of whithering syndrome. J. Invertebr. Pathol.
20, 867–874.
89, 219–231.
Moore, J., Juhasz, C., Robbins, T., Vilchis, I., 2009. Green abalone, Haliotis fulgens
Díaz, F., Río-Portilla, M., Sierra, E., Aguilar, M., Re-Araujo, D., 2000. Preferred
infected with the agent of withering syndrome do not express disease signs
temperature and critical thermal maxima of red abalone Haliotis rufescens. J.
under a temperature regime permissive for red abalone, Haliotis rufescens. Mar.
Therm. Biol. 25, 257–261.
Biol. 156, 2325–2330.
Díaz, F., Medina, Z., Valdez, G., Valenzuela, F., 2006. Thermal preference and
Navarro, J., Leiva, G., Gallardo, C., Varela, C., 2002. Influence of diet and temperature
tolerance of green abalone Haliotis fulgens (Philippi, 1845) and pink abalone
on physiological energetics of Chorus giganteus (Gatropoda: Muricidae) during
Haliotis Corrugata (gray, 1828). Aquac. Res. 37, pp. 877–844.
reproductive conditioning. NZ J. Mar. Freshwat. Res. 36, 321–332.
Elliott, J., Davidson, W., 1975. Energy equivalents of oxygen consumption in animal
Newell, R., 1985. Physiological effects of the MSX parasite Haplosporidium Nelsoni
energetics. Oecologia 19, 195–201.
(Haskin, Stauber and Mackin) on the American oyster, Crassostrea virginica. J.
Flores, A., Guitierrez, A., Ellwanger, A., Searcy-Bernal, R., 2007. Development and
Shellfish Res. 5, 91–95.
current status of abalone aquaculture in Chile. J. Shellfish Res. 26 (3), 705–711.
Peck, L., Culle, Y., Helm, M., 1987. A laboratory energy budget for the ormer Haliotis
Flye-Sainte-Marie, J., Pouvreau, S., Paillard, C., Jean, F., 2007. Impact of brown ring
tuberculata. J. Exp. Mar. Biol. Ecol. 106, 103–123.
disease on the energy budget of the Manila clam Ruditapes philippinarum. J. Exp.
Ricker, W., 1968. Methods of Assessment of Fish Production in Fresh Water.
Mar. Biol. Ecol. 349, 378–389.
Blackwell L.B.P., 313 pp.
Friedman, C., Thompson, M., Chun, C., Haaker, P., Hedrick, R., 1997. Withering
Rosenblum, M., Viant, B., Braid, J., Moore, J., Friedman, C., Tjeerdema, R., 2005.
syndrome of the black abalone Haliotis cracherodii (Leach): water temperature,
Characterizing the metabolic actions of natural stresses in the california red
food availability, and parasites as possible causes. J. Shellfish Res. 16, 403–411.
abalone, Haliotis rufescens using 1H NMR metabolomics. Metabolomics 1, 199–
Friedman, C., Biggs, W., Shields, J., Hedrick, R., 2002. Transmission of withering
209.
syndrome in black abalone Haliotis cracherodii Leach. J. Shellfish Res. 21, 817–
Shaw, L., Battle, I., 1957. The gross microscopic anatomy of the digestive tract of the
824.
oyster Crassostrea virginica (Gmelin). Can. J. Zool. 35, 325–347.
García-Esquivel, Z., Montes-Magallón, S., González-Gómez, M., 2007. Effect of
Solorzano, L., 1969. Determination of ammonia in waters by the phenolhypochlorite
temperature and photoperiod on the growth, feed consumption, and
method. Limnol. Oceanogr. 14, 799–801.
biochemical content of juvenile green abalone, Haliotis fulgens, fed on a
Thompson, R., Bayne, L., 1974. Some relationships between growth, metabolism and
balanced diet. Aquaculture 262, 129–141.
food in the mussel Mytilus edulis. Mar. Biol. 27, 317–326.
Gardner, G., Harshbarger, J., Lake, J., Sawyer, T., Price, K., Stephenson, M., Haaker, P.,
Thompson, J., Higgins, D., Gibson, T., 1994. ClustalW: improving the sensitivity of
Togstad, H., 1995. Association of prokaryotes with symptomatic appearance of
progressive multiple sequence alignment through sequence weighting,
withering syndrome in black abalone Haliotis cracherodii. J. Invertebr. Pathol. 66,
position-specific gap penalties and weight matrix choice. Nucleic. Acids. Res.
111–120.
22, 4673–4680.
González, G., Brokordt, K., Winkler, F., 2010. Repeatability of physiological traits in
Vilchis, L., Tegner, M., Moore, J., Friedman, C., Riser, K., Robbins, T., Dayton, P., 2005.
juvenile Pacific abalone, Haliotis discus hannai. Mar. Biol. 157, 2195–2203.
Ocean warming effects on growth, reproduction, and survivorship of southern
Gordon, R., Cook, P., 2001. Word abalone supply, markets and pricing: historical,
California abalone. Ecol. Appl. 15, 469–480.
current and future. J. Shellfish Res. 20, 567–570.
Villalba, A., Reece, K., Camino Ordás, M., Casas, S., Figueras, A., 2004. Perkinsosis in
Jobling, M., 1981. Temperature tolerance and the final preferendum-rapid methods
molluscs: a review. Aquat. Liv. Resour. 17, 411–432.
for the assessment of optimum growth temperature. J. Fish. Biol. 19, 439–455.
Wetchateng, T., Friedman, C., Wight, N., Lee, P., Teng, P., Sriurairattana, S.,
Kennedy, V., Newell, R., Krantz, G., Otto, S., 1995. Reproductive capacity of the
Wongprasert, K., Withyachumnarnkul, B., 2010. Withering syndrome in the
eastern oyster Crassostrea virginica infected with the parasite Perkinsus marinus.
abalone Haliotis diversicolor supertexta. Dis. Aquat. Organ. 90, 69–76.
Dis. Aquat. Org. 23, 135–144.
Winkler, L., 1888. A Vízben Feloldott Oxigén Meghatározása. (The Determination of
Kismohandaka, G., Friedman, C., Roberts, W., Hedrick, R., Crosby, M., 1993.
Dissolved Oxygen in Water.) PhD thesis, Pázmány Péter University, Budapest.
Investigation of physiological parameters of black abalone with withering
Xusheng, G., Yongfeng, L., Yongxiang, L., Jun, L., 1990. Influence of temperature on
syndrome. J. Shellfish Res. 12, 131–132.
feeding and growth of the young abalone. Oceanol. Limnol. Sin. 21, 20–26.
Lamares, M., Wing, S., 2001. Calorific content of New Zealand marine macrophytes.
J. Mar. Freshwat. Res. 35, 335–341.