The prebiotic inulin increases the phenoloxidase activity and reduces the prevalence

of WSSV in whiteleg shrimp (Litopenaeus vannamei) cultured under

laboratory conditions
Antonio Luna-Gonzlez , Judith C. Almaraz-Salas, Jess A. Fierro-Coronado, Ma. del Carmen Flores-Miranda,
Hctor A. Gonzlez-Ocampo, Viridiana Peraza-Gmez
Centro Interdisciplinario de Investigacin para el Desarrollo Integral Regional-Instituto Politcnico Nacional, Unidad Sinaloa, Sinaloa, Mexico
a b s t r a c t a r t i c l e i n f o
Article history:
Received 17 May 2012
Received in revised form 18 July 2012
Accepted 19 July 2012
Available online 27 July 2012
Keywords:
Inulin
Prebiotic
Litopenaeus vannamei
WSSV
Phenoloxidase
The effect of inulin on growth performance, survival, lactic acid bacteria (LAB) in the gut, WSSV prevalence, and
immune response of Litopenaeus vannamei was evaluated under laboratory conditions. Inulin was sprayed onto
feed at 0, 1.25, 2.5, 5.0, and 10 g kg feed
1
. Two bioassays, performed with treatments in triplicate, were
conducted for 62 and 73 days, respectively. Feed supplemented with inulin did not improve growth, survival,
and LAB in shrimp. However, inulin decreased the prevalence of WSSV in treated shrimp. The prebiotic signi-
cantly increased the phenoloxidase activity, but hemocyte number was not affected. Inulin increases the
phenoloxidase activity in L. vannamei and, at concentrations of 2.5 and 5.0 g kg feed
1
, is a good feed additive
against WSSV in shrimp with low viral load.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Shrimp aquaculture is an important worldwide industry. However,
since several years ago shrimp farming has been threatened by diseases
that have affected its production performance due to mismanagement
and the lack of biosecurity protocols. Viral diseases, such as the white
spot syndrome virus (WSSV), can cause severe mortalities in cultured
shrimps (Chou et al., 1995; Leu et al., 2009; Lo et al., 2003). In Mexico,
the states of Sonora and Sinaloa, located inthe northwest of the country,
are the most important whiteleg shrimp producers; however, inthe last
years important losses have occurred due to WSSV (CONAPESCA, 2010;
Peinado-Guevara and Lpez-Meyer, 2006).
Traditionally, to successfully restrict pathogen infection, farmers
apply basic practices of good management and use chemotherapy
(antibiotics) (Subasinghe and Barg, 1998). Shrimp cannot be vacci-
nated, thus, antibiotics are currently used; however, these chemicals
have been gradually prohibited due to the potential development of
antibiotic-resistant bacteria, presence of antibiotic residues in sea-
food, environmental impact, and suppression of the aquatic animals'
immune system (Li et al., 2007; Zhou et al., 2007). An alternative to
the use of antibiotics as growth promoters is to feed natural origin
additives such as probiotics, prebiotics, immunostimulants, and me-
dicinal plants (Partida-Arangure, unpublished data).
Immunostimulants are aimed at enhancing the non-specic de-
fense mechanisms in animals. A number of different biological and
synthetic compounds have been found to enhance the non-specic
defense system in animals, including shrimp (Song and Sung, 1990;
Sung et al., 1991).
Prebiotics are non digestible polysaccharides added to feed that bene-
cially affect the host by selectively stimulating the growth of and/or ac-
tivating the metabolism of one or a limited number of health-promoting
bacteria inthe intestinal tract, thus improving the host's intestinal balance
(Gibson and Roberfroid, 1995; Manning and Gibson, 2004). The prebi-
otics include fructooligosaccharides (FOS), transgalactooligosaccharides
(TOS), mannanoligosaccharides (MOS), lactose, and inulin (Teitelbaum
and Walker, 2002; Vulevic et al., 2004). Inulin and its derivates
(oligofructose, fructooligosaccharides) are generally known as fructans
and are basically constituted by linear chains of fructose (Madrigal and
Sangronis, 2007). Several inulin types occur in nature and they differ
in their degree of polymerization and molecular weight, depending on
the source, the harvest time, and processing conditions (Vijn and
Smeekens, 1999). Diets supplemented withFOS have beenshownto im-
prove the immunity and growth rate of aquatic animals such as
soft-shell turtle (Ji et al., 2004), turbot larvae (Mahious et al., 2006),
and white shrimp (Li et al., 2007; Zhou et al., 2007).
Shrimp possess an innate immune system. The hemocytes and plas-
matic molecules are key elements against pathogens. Hemocytes play a
central role in the immune response of shrimp, which rely mainly on
Aquaculture 362363 (2012) 2832
Corresponding author at: Centro Interdisciplinario de Investigacin para el
Desarrollo Integral Regional (Unidad Sinaloa), Boulevard Juan de Dios Btiz Paredes
250, Guasave, Sinaloa 81101, Mexico. Tel./fax: +52 687 87 2 96 26.
E-mail address: aluna@ipn.mx (A. Luna-Gonzlez).
0044-8486/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2012.07.022
Contents lists available at SciVerse ScienceDirect
Aquaculture
j our nal homepage: www. el sevi er . com/ l ocat e/ aqua- onl i ne
phagocytosis, melanization through the activation of the proPO cas-
cade, encapsulation, cytotoxicity, and hemolymph clotting mechanism
(Cerenius et al., 2008; Sritunyalucksana et al., 1999). Humoral defense
factors, such as agglutinins, clotting proteins, lisosomal hydrolytic en-
zymes (proteases, glycosidases, lipases, phosphatases), and antimicro-
bial peptides (penaedins) are released upon lysis of hemocytes, which
is induced by microbial surface antigens, such as peptidoglycans, lipo-
polysaccharides (LPS), and b-1,3-glucans (Chisholm and Smith, 1995;
Destoumieux et al., 2000; Muta and Iwanaga, 1996; Sderhll et al.,
1994).
The aim of this study was to evaluate the effect of the prebiotic in-
ulin on growth, survival, immune system, and prevalence of WSSV in
Litopenaeus vannamei cultured under laboratory conditions.
2. Materials and methods
2.1. Animals
Two batches of 150 and120 apparently healthy shrimp, basedonvis-
ible features, were collected from a commercial farm (Acucola Cuate
Machado, Guasave, Sinaloa, Mexico) and immediately transported to
the lab facilities of CIIDIR Sinaloa in a plastic container (250 L) provided
withsea water and aeration. The collectedshrimp hadnosigns of WSSV,
IHHNV, and/or bacterial infections. However, farmers specied that
shrimp had WSSV.
2.2. Shrimp acclimation to culture conditions
The healthy shrimp selection was done based on visible features.
Shrimpwere acclimatedto culture conditions for 3 days in120-L indoor
plastic tanks containing 80 L of ltered (20 m) sea water (3435)
and constant aeration in groups of 10 organisms per tank. Shrimp
were fed twice daily at 09:00 and 17:00 h with commercial feed
(Purina, Mexico, 35% protein). Feeding ration was 7% of mean body
weight. Half of the water was exchanged at day three. Uneaten food
and waste matter were removed daily before feeding.
2.3. Preparation of experimental diets with inulin
The concentrations of the prebiotic in feed were based on the
works of Li et al. (2007) and Zhou et al. (2007). Inulin from blue
agave (Agave tequilana, IIDEAL, S.A. de C.V., Guadalajara, Jalisco,
Mexico) was diluted in the adhesive and feed attractant Dry Oil
(DO, Innovaciones Acucolas, Mexico) and then sprayed onto pel-
lets in all treatments (including control group). Feed, prepared
for 10 days, was dried at room temperature for 4 h and then stored
at 4 C.
2.4. Experimental design
Two bioassays were conducted to evaluate the effect of feed sup-
plemented with inulin on cultured shrimp. Animals were maintained in
an outdoor culture system in 120-L plastic tanks with 80 L ltered
(20 m) seawater and constant aeration. Each treatment had three repli-
cates with 10 shrimp per tank. Shrimp were fed with commercial feed
(Purina, 35% protein) twice daily at 09:00 and 16:00 h. Initially, animals
were fed 7% of the mean body weight and adjusted thereafter according
to the feeding response in each tank. Uneaten food and waste matter
were removed every 3 days before feeding and 50% of the water was
exchanged.
Values of pH (HI 98127 pHep, Hanna Instruments, Woonsocket, RI,
USA), salinity (Refractometer W/ATC 300011, Sper Scientic, Scottsdale,
AZ, USA), dissolved oxygen, and temperature (YSI model 55 oxygen
meter, YellowSpring Instruments, YellowSprings, OH, USA) were moni-
tored every 3 days. At the beginning and at the end of each bioassay.
nitrites, nitrates, and ammonium were determined (Strickland and
Parsons, 1972).
The rst bioassay was conducted for 62 days with shrimp weighing
1.10.08 g. The distribution of shrimp in the tanks was at random. The
experiment was conducted as a completely randomized design with
ve treatments: (I) shrimp fed with commercial feed (control group);
(II) shrimp fed with commercial feed+inulin (1.25 g kg feed
1
); (III)
shrimp fed with commercial feed+inulin (2.5 g kg feed
1
); (IV) shrimp
fed with commercial feed+inulin (5.0 g kg feed
1
); (V) shrimp fed
with commercial feed+inulin (10 g kg feed
1
). At the end of the bioas-
say, survival and weight were determined. In addition, 12 shrimp per
treatment were analyzed individually for WSSV by single or nested PCR.
Negative samples were tested with an internal control that amplied a
298 bp segment of shrimp GAPDH DNA by one-step PCR.
For the count of presumptive lactic acid bacteria (LAB), the gut,
immediately posterior to the hepatopancreas, of three shrimp per
tank was aseptically removed, weighed, and placed in a precooled
(4 C) Eppendorf tube with 500 L of sterile saline (2.5% NaCl) solu-
tion. The sample was homogenized using a Pellet Pestle motor (Kontes,
NY, USA) at 4 C. One microliter of homogenized sample was spread on
MRS agar plates with 2% NaCl and 200 mg L
1
aniline blue. The plates
(nine per treatment) were incubatedat 32 C for 24 h. The presumptive
LAB CFU were counted at 48 h and expressed as CFU g gut
1
.
The second bioassay was conducted for 73 days with shrimp
weighing 1.090.07 g. The distribution of shrimp in the tanks was
at random. The experiment was conducted as a completely random-
ized design with two treatments: (I) shrimp fed with commercial
feed (control group); (II) shrimp fed with commercial feed+inulin
(2.5 g kg feed
1
). At the end of the bioassay, survival and weight
were determined. Hemolymph was extracted for immune system
analysis. At the end of the bioassay, shrimp fed with inulin were not
analyzed for WSSV because shrimp of the control group were WSSV
negative.
The specifc growth rate (SGR) was determined using the follow-
ing equation (Ziaei-Nejad et al., 2006):
SGR lnW
t
lnW
0
100=t
where t is the culture period in days, lnW
0
is the natural logarithm of
the weight of the shrimp at the beginning of the experiment and lnW
t
is the natural logarithm of the weight of the shrimp at day t (W
0
and
W
t
are in grams).
2.5. Prevalence of WSSV
Twelve shrimp per treatment (four per tank) were used to deter-
mine the prevalence of WSSV. Viral detection was performed by sin-
gle and nested PCR, using the primers WSSV out-1/WSSV out-2 and
WSSV in-1/WSSV in-2 (Kimura et al., 1996), which amplied genome
fragments of 982 and 570 bp, respectively. Negative samples were
tested with an internal control that amplied a 298 bp segment of
shrimp GAPDH DNA using the primers GAPDH298F and GAPDH298R
by one-step PCR (Tang and Lightner, 2001).
2.6. Hemolymph collection and total hemocyte count (THC)
Hemolymph was sampled from 12 intermolt shrimp per treat-
ment and THC was determined. Hemolymph (100 L) of individual
shrimp was withdrawn from the pleopod base of the rst abdominal
segment with a sterile 1-mL syringe (25 G13 mm needle). Before
hemolymph extraction, the syringe was loaded with 300 L of a
precooled (4 C) solution (SIC-EDTA, Na
2
) (450 mM NaCl, 10 mM
KCl, 10 mM hepes, and 10 mM EDTA, Na
2
at pH 7.3) used as an anti-
coagulant (Vargas-Albores et al., 1993). Fifty microliters of the
anticoagulant-hemolymph mixture was diluted in 150 L of formal-
dehyde (%) and then 15 L were placed on a hemocytometer (Neubauer)
29 A. Luna-Gonzlez et al. / Aquaculture 362363 (2012) 2832
to determine the THC using a compound microscope. The remainder of
the hemolymph was stored individually in Eppendorf tubes and kept on
ice for separation of plasma and hemocytes.
2.7. Separation of plasma and hemocytes
Samples of hemolymph were immediately centrifuged at 800 g for
5 min at 4 C andthe plasma was frozen at 80 C. The hemocyte pellet
was re-suspended and washed once in 1-mL precooled anticoagulant
solution by centrifugation at 800 g for 10 min at 4 C. Finally, the
hemocytes were re-suspended in 300 L cacodylate buffer (10 mM, pH
7). Individual samples were centrifuged at 14000 g for 10 min at 4 C
and the hemocyte lysate supernatant (HLS) was used immediately to
run the immunological analysis or stored at 80 C.
2.8. Phenoloxidase activity (PO) assay in plasma and HLS
PO activity was measured spectrophotometrically by recording the
formation of dopachrome produced from L-dihydroxyphenylalanine
(L-DOPA) following the procedures of Hernndez-Lpez et al. (1996).
For plasma, an aliquot of 50 L plus 50 L cacodylate buffer (10 mM,
pH7) and 50 L L-DOPA (3 mg mL
1
in distilled water) was incubated
at 37 C for 10 min, followed by 800 L cacodylate buffer. The optical
density at 492 nm was measured using a Thermo Spectronic Genesys
2 spectrophotometer (Thermo Scientic, Waltham, MA, USA). L-DOPA
plus cacodylate buffer was used as negative control. For HLS, an aliquot
of 50 L was incubated with 50 L trypsin (0.1 mg mL
1
), whichserved
as anactivator, for 30 min at 37 C; 50 L of L-DOPAwas then added and
incubated for 10 min at 37 C, followed by 800 L of cacodylate buffer.
The rest was as above. Activity was expressed as the variations in absor-
bance after 10 min.
2.9. Protein determination
In the second bioassay, protein concentration in plasma and HLS was
determined according to the method described by Bradford (1976), with
bovine serum albumin (BSA) from Sigma as standard. In plasma, protein
concentrationwas 14624 mg mL
1
in treatment I (control group) and
115.5938 mg mL
1
in treatment II. In HLS, protein concentration was
0.240.06 mg mL
1
and 0.240.05 mg mL
1
, respectively.
2.10. Statistical analysis
One-way analysis of variance (ANOVA) using the F test was applied
to examine the differences in total hemocytes count and survival (%)
among treatments. Survival data were arcsine transformed according
to Daniel (1997). Where signicant ANOVA differences were found, a
Tukey's HSD test was used to identify the nature of these differences
at pb0.05.
3. Results
3.1. Effect of inulinon survival, WSSV prevalence, SGR, and LAB (bioassay 1)
Shrimp survival (Table 1) was high (8010 to 965.7%) in all treat-
ments and no signicant differences were found among the treatments
(p>0.05). WSSV prevalence (Table 1) in treatment I was 58%; in treat-
ment II, 41.7%; in treatment III, 16.6%; in treatment IV, 16%; in treatment
V, 41.7%. Results showed that inulin reduces WSSV prevalence, especially
at concentrations of 2.5 and 5 g inulin kg feed
1
. Results (Table 1)
showedvalues of SGRfrom2.90.2to 3.00.0(% day
1
). Experimental
shrimp were not affected by the treatments with inulin, because growth
showed no signicant differences among treatments with inulin and con-
trol (p>0.05). LAB in shrimp gut fed with inulin did not show a clear
trend (Table 1).
3.2. Physicochemical parameters
During the experiment, water temperature ranged from 21.81.7
to 221.7 C, dissolved oxygen from 4.50.2 to 4.80.5 mg L
1
, sa-
linity from343.3 to 363.5, pHfrom7.80.2 to 8.00.2, nitrites
from0.030.01 to 0.070.05 mg L
1
, nitrates ranged from0.110.1
to 0.150.1 mg L
1
, and ammonium from 0.080.0 to 0.18
0.2 mg L
1
. According to these results, the physicochemical parame-
ters were within acceptable ranges, with the exception of temperature,
which was slightly below the accepted range (2330 C) (Boyd and
Tucker, 1998).
3.3. Effect of inulin on shrimp survival, SGR, and THC (bioassay 2)
No signicant differences were found between treatments I and II
(p>0.05) in shrimp survival, SGR, and THC (Table 2).
3.4. PO in HLS (proPO) and plasma
PO activity (Abs 492 nm) in hemocytes (proPO) was 0.410.01 in
control group and 0.800.17 in treatment II with inulin. PO activity
in plasma was 0.160.01 in control group and 0.270.03 in treatment
II with inulin. Inulin signicantly increased the PO activity (pb0.05) in
hemocytes (proPO) and plasma as compared with the control group
without inulin (Fig. 1).
3.5. Physicochemical parameters
During the experiment, the water temperature ranged from
27.61.56 to 27.61.59 C, dissolved oxygen from 7.30.29 to
7.30.32 mg L
1
, salinity from 32.61.81 to 35.03.80, pH
from 7.80.30 to 8.00.04, nitrites from 0.030.00 to 0.05
0.05, nitrates from0.640.25 to 0.810.06 32 mg L
1
, and ammo-
nium from 0.710.03 to 0.770.02 mg L
1
. According to these re-
sults, the physicochemical parameters were within acceptable ranges
(Boyd and Tucker, 1998).
4. Discussion
Control strategies against shrimp diseases are necessary (Li-Shi et
al., 2007). In accordance with this point of view, the present study
Table 1
Survival, WSSV prevalence, SGR, and LAB in shrimp fed commercial feed with inulin.
Treatments Shrimp survival
(%)
WSSV prevalence
(%)
SGR
(% day
1
)
LAB
(CFU g gut
1
)
I 9010 58.0 2.90.2 5435
II 965.7 41.7 2.90.2 3417
III 935.7 16.6 2.90.0 29046
IV 9010 16.0 3.00.0 33.841
V 8010 41.7 3.00.0 218.732
(I) Shrimp fed with commercial feed (control group); (II) shrimp fed with commercial
feed+inulin (1.25 g kg feed
1
); (III) shrimp fed with commercial feed+inulin
(2.5 g kg feed
1
); (IV) shrimp fed with commercial feed+inulin (5.0 g kg feed
1
);
(V) shrimp fed with commercial feed+inulin (10 g kg feed
1
). Survival, SGR, and
LAB data represent the meanSD. WSSV, white spot syndrome virus.
Table 2
Survival, SGR, and THC in shrimp fed commercial feed with inulin.
Treatments Shrimp survival (%) SGR (% day
1
) Total hemocyte
count (cells mL
1
)
I 965.7 3.090.08 910
6
II 1000 2.930.07 1010
6
(I) Shrimp fed with commercial feed (control group); (II) shrimp fed with commercial
feed+inulin (2.5 g kg feed
1
). The survival and SGR data represent the meanSD.
30 A. Luna-Gonzlez et al. / Aquaculture 362363 (2012) 2832
was carried out to investigate whether oral administration of prebiotics
is capable of protecting L. vannamei against WSSV. Prebiotics have been
recognized for increasing growth rate, improve immune response, as
well as change the community of gastrointestinal microbiota in cul-
tured animals (Li et al., 2007; Yousean and Sheikholeslami, 2009;
Zhou et al., 2007).
Inthe rst bioassay of this work, shrimpfedwithinulindidnot show
a signicant increase in weight and survival compared to the control
group. These results are consistent with those obtained by Li et al.
(2007) who found no signicant increase in weight and survival of
L. vannamei fed with fructooligosaccharides in the diet (0.025, 0.0500,
0.075, 0.100, 0.200, 0.400, and 0.800%). In contrast with our study,
Zhou et al. (2007) found a signicant increase in growth in L. vannamei
fed with fructooligosaccharides in the diet (0, 0.4, 0.8, 1.2, and
1.6 g kg
1
) at concentrations of 0.4 to 1.6 g kg
1
, although their best re-
sult was at 0.4 g kg
1
.
LAB in shrimp gut did not show a clear trend with the tested con-
centrations of inulin. However, in the work of Li et al. (2007) the addi-
tion of fructooligosaccharides to the diet increased the number of
bacteria (uncultured microbes, Alkalibacillus sp., Micrococcus sp., and
Roseobacter sp.) in the shrimp gut. In the same way, in the work of
Zhou et al. (2007), fructooligosaccharides increased the number of
bacteria (Vibrio parahaemolyticus, Aeromonas hydrophila, Lactobacillus
sp., and Streptococcus faecalis) in the shrimp gut. It is important to
note that inulin tested in this study is a fructan from blue agave (Agave
tequilana) and consists of a linear and linear and branched mixture of
(21) and (26) linkages with a DP (degrees of polymerization)
range of 332 (Lpez et al., 2003) whereas fructooligosaccharides
with DP 39 (average DP 4.5) are produced during the process of
chemical degradation or controlled enzymatic hydrolysis of inulin by
endoglycosidases (Roberfroid et al., 1998).
Inulin in the diet decreased the prevalence of WSSV in shrimp with
lowviral load and apparently healthy. There are no reports on what has
beenfound inshrimpor other crustaceans or invertebrates; although, in
human medicine, in vitro studies have shown that the oligosaccharides
(OS) of breast milk have a similar structure to that of specic receptors
of bacteria, toxins, and viruses, which act as competitive receptors in in-
testinal epithelial cells of the host, preventing the adhesion of patho-
gens. It has been shown that the OS-2 inhibits the binding ligands of
host cells with Campylobacter jejuni, Calicivirus, ST enterotoxin of
Escherichia coli, Streptococcus pneumoniae, Haemophilus inuenzae, and
Helicobacter pylori (Vitoria-Miana, 2007).
In the second bioassay, inulin did not affect weight and survival of
shrimp fed with the prebiotic as compared with the control group.
Neither did we nd an increase in THC, as observed in the work of Li
et al. (2007). Hemocytes are the rst line of defense in invertebrates
(Cerenius et al., 2008). However, inulin increased the POactivity in plas-
ma and HLS in shrimp fed with inulin, similar to the results of Li et al.
(2007), who mentioned that the addition of fructooligosaccharides sig-
nicantly increased PO activity in shrimp fed 0.1 and 0.8% of scFOS.
The prophenoloxidase cascade is a key element of the shrimp humoral
response. In L. vannamei, proPO is involved in immune defense against
Vibrio alginolyticus (Yeh et al., 2009) but it is inhibited by WSSVinfection
(Ai et al., 2008).
5. Conclusion
Inulin increases the PO activity on L. vannamei. This study is the rst
report to show that a prebiotic, inulin, reduces the WSSV prevalence in
shrimp with low viral load.
Acknowledgments
Authors are grateful to Consejo Estatal de Ciencia y Tecnologa del Estado
de Sinaloa (CECyT-Sinaloa) and Secretara de Investigacin y Posgrado del
Instituto Politcnico Nacional (SIP-IPN) for nancial support. JudithCristina
Almaraz Salas acknowledges CONACYT Mexico and SIP-IPN for the M.S.
grants.
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Fig. 1. Phenoloxidase activityinhemocyte lysate supernatant (proPO) andplasma. (I) Shrimp
fed with commercial feed (control group); (II) shrimp fed with commercial feed+inulin
(2.5 g kg feed
1
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