Prebiotics_2012 Gonzalles et al

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The prebiotic inulin increases the phenoloxidase activity and reduces the prevalenceof WSSV in whiteleg shrimp (Litopenaeus vannamei) cultured underlaboratory 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 ta 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 ofLitopenaeus vannameiwas evaluated under laboratory conditions. Inulin was sprayed onto

feed at 0, 1.25, 2.5, 5.0, and 10 g kg feed1. 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 inL. vannameiand, at concentrations of 2.5 and 5.0 g kg feed1, 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,

thestatesof Sonoraand Sinaloa, located in thenorthwestof thecountry,

are the most important whiteleg shrimp producers; however, in the 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 in theintestinaltract, thusimprovingthe 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 with FOShave been shown to 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:[email protected](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 o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a q u a - o n l i n e
http://dx.doi.org/10.1016/j.aquaculture.2012.07.022http://dx.doi.org/10.1016/j.aquaculture.2012.07.022http://dx.doi.org/10.1016/j.aquaculture.2012.07.022http://dx.doi.org/10.1016/j.aquaculture.2012.07.022http://dx.doi.org/10.1016/j.aquaculture.2012.07.022mailto:[email protected]://dx.doi.org/10.1016/j.aquaculture.2012.07.022http://www.sciencedirect.com/science/journal/00448486http://www.sciencedirect.com/science/journal/00448486http://dx.doi.org/10.1016/j.aquaculture.2012.07.022mailto:[email protected]://dx.doi.org/10.1016/j.aquaculture.2012.07.022


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phagocytosis, melanization through the activation of the proPO cas-

cade, encapsulation, cytotoxicity, and hemolymph clottingmechanism

(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 vannameicultured under laboratory conditions.

2. Materials and methods

2.1. Animals

Twobatches of 150and 120apparently healthy shrimp, based on vis-

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

with seawaterand aeration.The collectedshrimp hadno signs 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 acclimated to cultureconditions for3 days in 120-L indoor

plastic tanks containing 80 L ofltered (20m) 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 ofLi et al. (2007)andZhou et al. (2007). Inulin from blue

agave (Agave tequil ana, 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

(20m) 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, Yellow Spring Instruments, Yellow Springs, 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).

Therst 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 feed1); (III)

shrimp fed with commercial feed+ inulin (2.5 g kg feed1); (IV) shrimp

fed with commercial feed+ inulin (5.0 g kg feed

1

); (V) shrimp fedwith commercial feed+ inulin (10 g kg feed1).At the end ofthebioas-

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 wasspread on

MRS agar plates with 2% NaCl and 200 mg L1 aniline blue. The plates

(nine per treatment) were incubated at 32 Cfor 24 h. Thepresumptive

LAB CFU were counted at 48 h and expressed as CFU g gut1.

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 feed1). 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 lnWtlnW0 100=t

wheretis the culture period in days, lnW0is the natural logarithm of

the weight of the shrimp at the beginning of the experiment and lnWtis the natural logarithm of the weight of the shrimp at day t(W0and

Wtare 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 GAPDH298Rby 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, Na2) (450 mM NaCl, 10 mM

KCl, 10 mM hepes, and 10 mM EDTA, Na2at 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 15L were placed on a hemocytometer (Neubauer)

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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 800gfor

5 min at 4 Cand the plasma was frozenat80 C. Thehemocyte pellet

was re-suspended and washed once in 1-mL precooled anticoagulantsolution by centrifugation at 800g 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 gfor 10 min at 4 C

and the hemocyte lysate supernatant (HLS) was used immediately to

run the immunological analysis or stored at80 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 ofHernndez-Lpez et al. (1996).

For plasma, an aliquot of 50L plus50L cacodylatebuffer (10 mM,

pH 7) and 50LL-DOPA (3 mg mL1 in distilled water) was incubated

at 37 C for 10 min, followed by 800 L cacodylate buffer. The opticaldensity 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

of50L wasincubated with 50L trypsin (0.1 mg mL1), which served

as anactivator,for 30 min at 37 C; 50L ofL-DOPA was then added and

incubated for 10 min at 37 C, followed by 800L 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, proteinconcentration was 14624 mg mL1 in treatment I (control group) and

115.5938 mg mL1 in treatment II. In HLS, protein concentration was

0.240.06 mg mL1 and 0.240.05 mg mL1, respectively.

2.10. Statistical analysis

One-way analysis of variance(ANOVA) using the Ftest was applied

to examine the differences in total hemocytes count and survival (%)

among treatments. Survival data were arcsine transformed according

toDaniel (1997). Where signicant ANOVA differences were found, a

Tukey's HSD test was used to identify the nature of these differences

atpb0.05.

3. Results

3.1. Effect of inulin on survival, WSSVprevalence, SGR,and LAB (bioassay1)

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%. Resultsshowed that inulin reduces WSSV prevalence, especially

at concentrations of 2.5 and 5 g inulin kg feed1. Results (Table 1)

showed values of SGR from 2.9 0.2 to 3.0 0.0 (% day1). Experimental

shrimp were not affected by the treatments with inulin, because growth

showed no signicant differences among treatments with inulinand 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 L1, sa-

linity from 343.3 to 363.5, pH from 7.80.2 to 8.00.2, nitrites

from 0.030.01to 0.070.05 mg L1, nitrates ranged from 0.11 0.1

to 0.150.1 mg L1, and ammonium from 0.080.0 to 0.18

0.2 mg L1. 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 0.16 0.01 in control group and 0.270.03 in treatment

II with inulin. Inulin signicantly increased the PO activity (pb0.05) inhemocytes (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 L1, 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 from 0.64 0.25 to 0.81 0.06 32 mg L1, and ammo-

nium from 0.710.03 to 0.77 0.02 mg L1. 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

(% day1)

LAB

(CFU g gut1)

I 9010 58.0 2.9 0.2 5435

II 965.7 41.7 2.9 0.2 3417

III 935.7 16.6 2.9 0.0 29046

IV 9010 16.0 3.0 0.0 33.841

V 8010 41.7 3.0 0.0 218.732

(I) Shrimp fed with commercial feed (control group); (II) shrimp fed with commercial

feed+inulin (1.25 g kg feed1); (III) shrimp fed with commercial feed+inulin

(2.5 g kg feed1); (IV) shrimp fed with commercial feed+inulin (5.0 g kg feed1);

(V) shrimp fed with commercial feed+inulin (10 g kg feed1). 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 (% day1) T ot al hemocyte

count (cells mL1)

I 96 5.7 3.09 0.08 9 106

II 100 0 2.93 0.07 10 106

(I) Shrimp fed with commercial feed (control group); (II) shrimp fed with commercial

feed+inulin (2.5 g kg feed1

). The survival and SGR data represent the mean SD.

30 A. Luna-Gonzlez et al. / Aquaculture 362363 (2012) 2832


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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).

Intherst bioassay of this work, shrimpfed with inulindid notshow

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. vannameifed 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 inL. vannamei

fed with fructooligosaccharides in the diet (0, 0.4, 0.8, 1.2, and

1.6 g kg1) at concentrationsof 0.4to 1.6 g kg1, although their best re-

sult was at 0.4 g kg1.

LAB in shrimp gut did not show a clear trend with the tested con-

centrations of inulin. However, in the work ofLi et al. (2007) the addi-

tion of fructooligosaccharides to the diet increased the number of

bacteria (uncultured microbes, Alkalibacillussp.,Micrococcussp., and

Roseobactersp.) 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

low viral load and apparently healthy. There are no reports on what has

been found in shrimp or other crustaceans or invertebrates; although,in

human medicine,in vitrostudies 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 ofLi

et al. (2007). Hemocytes are the rst line of defense in invertebrates

(Cerenius et al., 2008). However, inulin increased the PO activity in plas-

ma and HLS in shrimp fed with inulin, similar to the results ofLi 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. InL. vannamei, proPO is involved in immune defense against

Vibrio alginolyticus(Yeh etal.,2009) but it is inhibitedby WSSV infection(Ai et al., 2008).

5. Conclusion

Inulin increases the PO activity on L. vannamei. This study istherst

report to show that a prebiotic, inulin, reduces the WSSV prevalence in

shrimp with low viral load.

Acknowledgments

Authors aregrateful to ConsejoEstatal de Ciencia y Tecnologa del Estado

de Sinaloa(CECyT-Sinaloa) andSecretara de Investigacin y Posgrado del

Instituto Politcnico Nacional (SIP-IPN) fornancial support. JudithCristina

Almaraz Salas acknowledges CONACYT Mexico and SIP-IPN for the M.S.grants.

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(2.5 g kg feed1). PO data represent the meanSD. Different letters indicate signicantdif-

ference (pb

0.05).

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