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bG MOS effect in the larval rearing of ounder 35Invest. Mar., Valparaso, 35(2): 35-43, 2007

Effect of the application of -glucans and mannan-oligosaccharides (GMOS) in an intensive larval rearing system ofParalichthys adspersus

(Paralichthydae)

Nicole Piaget1, Alonso Vega1,3, Alfonso Silva2 & Pedro Toledo2,31Departamento de Biologa Marina, Facultad de Ciencias del Mar

Universidad Catlica del Norte, Casilla 117, Coquimbo, Chile2Departamento de Acuicultura, Facultad de Ciencias del MarUniversidad Catlica del Norte, Casilla 117, Coquimbo, Chile

3CEAZA, Centro de Estudios Avanzados de Zonas ridas, Coquimbo, Chile

ABSTRACT. For successful rearing of the ounder Paralichthys adspersus, it is important to optimize growth and sur-vival in the early larval stages. Several authors indicate that the application of -glucans and mannan-oligosaccharides(G MOS) in rearing water should improve the larval health, diminishing the effects of physiological stress and physicaldamage that the aquaculture activities cause to the individuals. In order to evaluate the effect of G MOS on P. adspersusincorporation on P. adspersus larval survival and growth in intensive culture, experiments were carried out with six-dayspost-hatch larvae, which had only just begun to feed on live prey (rotifers), and fteen-day post-hatch larvae. Threetreatments were used, applying 5 mgL-1, 10 mgL-1, and 15 mgL-1 of G MOS to the rearing water during the rst vedays of the experiment and then comparing the results with a control. The results indicate that applications of 5 mgL-1of G MOS in the rearing water enhance larval survival and growth with respect to the control, whereas additions of 15

mgL-1

of G MOS suppressed both of these parameters. This effect increases for larvae that have recently absorbed theyolk sac. An histological analysis of the intestinal epithelium of the larvae suggests that G MOS promotes the expressionof monocytes (forerunner cells of macrophages) associated with the non-specic immune system of the sh.

Key words: Paralichthys adspersus, ounder, larval rearing, growth rate, survival, -glucan and mannan-oligosaccha-rides, immunestimulants.

Efecto de la aplicacin de -glucanos y manano-oligosacridos (G MOS) en unsistema de cultivo intensivo de larvas deParalichthys adspersus (Paralichthydae)

RESUMEN. Para el xito del cultivo del lenguado Paralichthys adspersus es importante optimizar el crecimiento y la

supervivencia en los primeros estados de desarrollo larval. Diversos autores sealan que la aplicacin de -glucanos ymanano-oligosacridos (G MOS) en el agua de cultivo debera mejorar la salud de las larvas, disminuyendo los efectosdel estrs siolgico y el dao fsico de los individuos causado por las actividades propias de la acuicultura. Con el objetivode evaluar el efecto de la incorporacin del G MOS en la supervivencia y el crecimiento de larvas de P. adspersus encultivos intensivos, se realizaron experimentos utilizando larvas de seis das post-eclosin que recin han comenzado laalimentacin con presas vivas (rotferos) y larvas de quince das post-eclosin. Tres tratamientos aplicando 5 mgL-1, 10mgL-1 y 15 mgL-1 de G MOS al agua de cultivo fueron contrastados durante los primeros cinco das de experimentacincon una condicin control. Los resultados indican que aplicar 5 mgL -1 de G MOS en el agua de cultivo aumenta lasupervivencia y el crecimiento de las larvas con respecto al control, mientras que 15 mgL-1 de G MOS tiene un efectosupresor en ambos parmetros poblacionales. Este efecto aumenta si se aplica en larvas que recin han absorbido el sacovitelino. El anlisis histolgico del epitelio intestinal de las larvas sugiere que el G MOS promueve la manifestacin demonocitos (clulas precursoras de macrfagos) asociados al sistema inmune no especco de los peces.

Palabras clave: Paralichthys adspersus, lenguado,cultivo larval, tasa de crecimiento, supervivencia, -glucanos ymanano-oligosacridos, inmunoestimulante.

Corresponding author: Nicole Piaget ([email protected])


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36 Investigaciones Marinas, Vol. 35(2) 2007

INTRODUCTION

Under intensive culture, the manipulation of indi-viduals specimens and the chemical and physicalconditions of the system cause physiological stress

and physical damage to the sh (Rottmann et al.,1992). Stress during sh larval rearing is intermittentand synergic, which amplies its negative effect andincreases the risk of mortality caused by opportunis-tic microorganisms or pathogenic agents (Skjermo& Vadstein, 1999; Ellis, 2001). The application ofprophylactic compounds such as -glucans and man-nan-oligosaccharides (G MOS) improves the healthof sh during the early development stages (pre-lar-va, larva, post-larva; sensu Silva, 2000), mainly bystimulating the non-specic immune system to create

defenses against viral, bacterial, and fungal attacks(Sakai, 1999; Raa, 2000). Moreover, applications ofcompounds containing -glucans (G) are coadju-vant, increasing the shs resistance to parasites andimproving the effectiveness of vaccines (Anderson,1992; Robertsen et al., 1994). The action of G in theintestine is similar to that of probiotics, stimulatingthe proliferation of benecial bacteria that assist theimmunological system, permitting decreased theuse of exogenous or greater effects from exogenousantibiotics. The mannan-oligosaccharides (MOS),on the other hand, absorb the mycotoxins found inthe nutrients commonly used in formulated diets(Vadstein,1997; Raa, 2000; Bergh et al., 2001; Pryoret al., 2003). Other compounds besides G MOSalso activate macrophages, including -interferon,peptides, proteins, and lipopolysaccharides (Sakai,1999; Raa, 2000; Jin & Xiao-ling, 2004; Bricknell& Dalmo, 2005; Kumari & Sahoo, 2006).

The application of G for prophylactic purposesin juvenile and adult farmed sh is done through

injections (intraperitoneal, pre-anal, or intravenous),or by incorporating these compounds into formulateddiets (Robertsen et al., 1994; Sakai, 1999). On theother hand, these compounds can be incorporatedthrough live prey or by applying them directly to therearing water as immersion baths during the earlystages of sh development (Skjermo & Vadstein,1999; Bergh et al., 2001; Skjermo & Bergh, 2004).The compounds that contain G act at the basal levelof the development of the immunological system andare soluble in sea water, facilitating their absorption

through the skin, gills, and mouth (Strand & Dalmo,1997; Dalmo et al., 2000; Raa, 2000). Nonetheless,although the protocols for administering the pro-phylactic compounds are effective transfer mecha-

nisms to strengthen the immunological system of thesh, the application times for activating and maintai-ning antibody levels during early development seemsto be species-specic (Bricknell & Dalmo, 2005).

Under larval rearing conditions, the individualsmust combat infectious diseases through the non-specic immune system (Ellis, 2001). This is be-cause the sh larvae have not developed a specicimmunological defense system, that is, the capacityto offer immune protection against pathogenic agents(Esteban et al., 1994; Bricknell & Dalmo, 2005).Thus, a preventive treatment (prophylactic) suchas the application of G MOS to the rearing watershould increase the survival of the larvae, strengthe-ning the non-specic immune system and, moreover,

minimizing the environmental problems associatedwith other alternative protocol such as vaccines and/or drug therapy (Anderson, 1992; Raa, 2000). Theconstant application of such protocols (e.g. vaccines,drugs) over time produces residuals that persist in theenvironment and are transmitted to other organisms,possibly reaching toxic levels. Furthermore, suchprotocols favor antibiotic resistance in the pathogensand affect the microbial activity responsible for thebreakdown of organic matter in the marine sediments(SERNAPESCA, 2005).

Different techniques have been used to determinewhether applied organic compounds improve larvalhealth, most noticeably biochemical analysis, assayswith pathogenic bacteria, histological cuts and, recen-tly, immunouorescence markers. The biochemicalanalysis are based on blood and/or serum samples(Kumari & Sahoo, 2006), which are very difcultto obtain from small individual larvae (Bricknell &Dalmo, 2005). As well, the assays with pathogenicbacteria have shown that the intrinsic factors of thefish (fitness, developmental stage, age) producegreater variability between replicates than betweentreatments, obscuring the effect of the applicationof the prophylactic compound (Bricknell & Dalmo,2005). Classic histological stains (nuclear, cellular),however, are similar in effectiveness and less costlythan immunoorescent markers for conrming theabsorption of these organic compounds in the shlarvae (Cousin et al., 1986; Esteban et al., 1994;Strand & Dalmo, 1997; Luizi et al., 1999; Ribeiro etal., 1999; Dalmo et al., 2000; Bergh et al., 2001).

In Peru and Chile, the ounder Paralichthysadspersus Steindachner, 1867 is an attractive speciesfor intensive culture (Silva, 2001; ngeles & Men-do, 2005). Nevertheless, one of the obstacles for its


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bG MOS effect in the larval rearing of ounder 37

culture, as with most potentially farmed sh species,is its high mortality during the rst larval stage. Infact, according to Silva (2000), the rst 21 days aftereclosion of the egg in P. adspersus rearing are critical;larval mortality nears 80% during this period. Suchmortality rates are frequently reported for larvalrearing of other Paralichthys species (Kuronuma& Fukusho, 1984; Bisbal & Bengtson, 1995; Silva,2001; Silva & Castell, 2005). Mortality in early andmore advanced sh development stages is recurrentlyassociated with diseases produced by infections fromopportunistic bacteria and the stress caused by mani-pulation during culture and the culture system itself(Miranda & Rojas, 1993; Skjermo & Vadstein, 1999;Ellis, 2001). For example, in P. adspersus, vibriosisis manifested when the sh present stress-relatedimmunedepression (Miranda & Rojas, 1996). Thus,we propose the hypothesis that the addition of GMOS should decrease mortality in intensive cultureofP. adspersus larvae. Accordingly, our objectiveis to evaluate survival and growth in the rst larvalstage of the farmed ounder given treatments withdifferent concentrations of G MOS in the rearingwater. Furthermore, we documented whether theG MOS compound strengthened the health of thelarvae, attempting to detect cells that characterizedthe non-specic immune system in the intestine.

MATERIALS AND METHODS

In order to evaluate the effect of G MOS in therearing water had on P. adspersus larvae, we carriedout two experiments in different time periods ofthe rst feeding (live prey) stage. This stage beginswhen the larvae have absorbed the yolk sac (4-5 dayspost-hatch) and begin to feed on rotifers (Brachionus

plicatilis); it ends between 20 and 25 days post-hatch,when the second stage of feeding (onArtemia) begins(Silva, 2001). In Experiment 1, 6-day post-eclosionlarvae were used and, whereas in Experiment 2,15-day post-hatch larvae were used. The -glucanand mannan-oligosaccharide compound used in theexperiments (called DP MOS G) was provided byDESPRO S.A. company (Desarrollo de Protenasde Chile S.A.). Used as a complement in the dietof farmed sh, this compound is an association of-glucans (46%), mannan-oligosaccharides (53%),

and cytoplasmatic content (1%). The G MOS wasobtained from yeast (Saccharomyces cerevisiae)through lysis and fractionation of the cell wall, witha particle size that uctuated between 0.5 and 10 .

Obtaining the larvae

The larvae were obtained from two spontaneousspawnings of the P. adspersus reproductive stock atthe Laboratorio de Peces, Universidad Catlica del

Norte, in September and October 2002. The fertilizedeggs were collected with a 400-m sieve placed inthe exterior drain tube of the tanks. The eggs wereltered, washed, and left to rest for approximately 20min in a 10-L recipient in order to select the viableeggs according to the protocol described by Silva &Castell (2005). The viable eggs, previously quan-tied and disinfected, were deposited in 100-L tanksand incubated for 54 to 62 h in ltered, sterilizedsea water with ultraviolet light (Silva & Castell,2005). The eclosed larvae were moved to a black,

cylindrical-conical 500-L tank with ltered sea water(salinity 34 0.5), aerated continuously, and kept atroom temperature (16-17C) until their extractionfor the experiments.

Experimental design

In Experiment 1, larvae of 4.0 0.3 mm standardlength (six-day post-hatch) were used and, in Ex-periment 2, larvae of 5.4 0.2 mm standard length(15-day post-hatch) were used. Both experimentswere carried out during the rst stage of feeding ofthe farmed larvae. In this stage, the larvae are fed withrotifers (Brachionus plicatilis; 5-10 rotifersmL-1)(Silva & Vlez, 2005). The rotifers, farmed in batchsystems, were fed yeast (Saccharomyces cerevisiae)and enriched with a mixture of microalgae (e.g.Isochrysis sp., Nannochloropsis sp.). Each experi-ment lasted ten days and, in both cases, the larvaewere exposed to treatments with concentrations of 5mgL-1, 10 mgL-1, and 15 mgL-1 G MOS throughimmersion (Tytler & Blaxter, 1988); a control group

was not treated with the compound. This concentra-tion range was previously used in other experimentsthat evaluated the effect of G-based prophylacticcompounds on fish larval survival and growth(Dalmo et al., 2000; Bergh et al., 2001; Skjermo &Bergh, 2004).

The treatments, with three replicates, were dis-tributed at random in 12 black, cylindrical, 30-Ltanks at a density of 30 larvaeL-1. The larval groupswere treated with G MOS dissolved in sea waterat concentrations of 5 mgL-1, 10 mgL-1, and 15

mgL-1, during the rst ve days of each experiment.The G MOS concentration for each treatment wasmaintained daily in experimental tank by absorbingthe water with a siphon and replacing it with the


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diluted compound in sea water. The sea water usedfor the G MOS dilutions was ltered at 1 m andsterilized with ultraviolet light (UV). On days 2,4, 6, 8, and 10 of the experiments, 30 larvae wereextracted from the experimental tanks with a siphon.The sampled larvae were excluded from the survivalanalyses. After each sampling, the volume of waterwas decreased proportionally in each tank in order tomaintain a constant density (30 larvaeL-1). In bothexperiments, all the utensils were duly washed anddisinfected.

In order to determine the effect of the G MOS onthe larvae, the percentage of survival was evaluatedalong with the growth rate and the manifestationof monocytes or macrophage precursor cells in the

larval intestines. The percentage of survival (S) wascalculated for each treatment using the equation S= (n

f/n

i) 100 (Downing & Litvak, 1999), where n

i

and nfare the initial and nal number of larvae. The

growth specic rate (Gs) was calculated for each

replicate of each treatment during the experimentalperiod with the equation G

s= ([ln

2- ln

1]/T

2- T

1)

100, where 1

is the standard length (mm) at timeT

1(Downing & Litvak, 1999). The standard length

() was measured from the extreme anterior pointof the upper mandible to the extreme posterior point

of the notochord (Downing & Litvak, 1999) usinga prole projector (Nikon V12). All the sampledlarvae were preserved in formaline diluted in seawater at 10% in black plastic bottles with labels forlater histological analyses (Muetn-Gmez et al.,2000; K. Lohrmann, pers. comm.).

The histological cuts ofP. adspersus larvae weredone at the intestinal level since the larvae had notyet developed a functional stomach. In this stage ofdevelopment, the anterior intestine is responsible forthe digestion processes and the posterior intestine forthe absorption processes (Ribeiro et al., 1999). Thelarvae collected for each treatment per sampling timewere dehydrated in an increasing battery of ethanolfor their later inclusion in parafn (Muetn-Gmezet al., 2000). The cuts of the anterior and posteriorsection of the larval intestines were 5 , using arotory microtome (Leitz 1512). The tissues werestained with Hematoxiline-eosine using the Entellanmounting medium and Hemacolor (Muetn-Gmezet al., 2000). All the cuts were checked under a light

microscope (maximum magnication 1000X) usingimmersion oil to evaluate the presence of monocyticcells that characterize the non-specic immune sys-tem in the larval intestines. The monocytic cells in

both intestinal sections were evaluated.

The survival (S) and growth (Gs) data for the

larvae in the experimental treatments were comparedusing analysis of varianza (ANOVA), prior to their

transformation to the arc-sine of the square root(Sokal & Rohlf, 1981). When the ANOVA showedsignicant differences, an a posteriori Tukey testwas performed to detect differences between thetreatment pairs. The effect of the application ofG MOS on the temporal increase in the standardlength was evaluated with an analysis of covariance(ANCOVA). Before carrying out the ANOVA andANCOVA, the normality of the data, the out-of-rangedata, and the homocedasticity of the variances wereproven through the Barlett test, the Lilliefors test,

and visual observation (Sokal & Rohlf, 1981) usingthe computer software SYSTAT 8.0.

RESULTS

Larval survival

The effect of G MOS on the survival ofP. adspersuslarvae was dependent on the concentration of dilutionapplied to the rearing water. No signicant differen-ces were found between the survival percentages of

six-day post-hatch individuals that received 5 mgL-1

G MOS in the larval rearing water (Experiment1) and the control group (0 mgL-1 G MOS; Fig.1). However, survival decreased signicantly (F

(3.8)

= 9.018, p < 0.05; Fig. 1) with the use of greaterconcentrations (10 and 15 mgL-1) of G MOS withrespect to the control group and the other treatment.For the 15-day post-hatch larvae (Experiment 2),the application of 5 mgL-1 of G MOS in the rea-ring water signicantly improved (F

(3.8)= 5.68, p 0.05) and signicantly

higher (F(3.8)

= 6.231, p < 0.05) than that observed forthe larvae treated with 10 and 15 mgL-1 G MOS(Fig. 3). Although the larvae treated with 5 mgL-1G MOS presented the greatest growth rates in bothexperiments, the compound was most effective forgrowth in the 6-day post-hatch larvae (Fig. 3).

Histological analysis

The histological analyses of the anterior andposterior intestines of the larvae stained with He-matoxiline-eosine and Hemacolor showed clearlydifferentiated monocytic cells, characterized by

well-dened nuclei (Fig. 4). The temporal follow-up of the treatments through histological analysesshowed a greater relative frequency of monocytes inthe groups treated with G MOS on the tenth day ofthe experiment than in the normal or control group.Likewise, a high variability was detected betweenthe histological cuts of the larval intestines given thesame treatment, making it impossible to statisticallyquantify and evaluate in which G MOS treatmentthe monocytes were more frequent. The presence ofmonocytic cell as a macrophage precursor is an evi-

dence that complements the results of the effect of theG MOS on the survival and growth ofP. adspersusthe larvae in intense culture systems.


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DISCUSSION

Among the administration protocols of -glucan-based compounds were successful in improving the

health of larvae reared in captivity, oral administra-tion was noteworthy. This administration protocol isuseful for rearing P. adspersus larvae when is givenduring the rst feeding stage with live prey dependson the concentration of G MOS applicated. Similarresults, using the same protocol, have been obtainedfor Hippoglossus hippoglossus and Scophthalmusmaximus larvae, as well as for other sh speciesraised in captivity (Dalmo et al., 2000; Bergh etal., 2001; Skjermo & Bergh, 2004). In our study,the application of 5 mgL-1 G MOS signicantly

increased survival and growth ofP. adspersus lar-vae, whereas concentrations of 15 mgL-1 G MOSsuppressed these population parameters. An excessof prophylactic compounds applied to Sparus auratasuppressed the non-specic immune response andhad negative consequences for the survival and de-velopment of the individuals (Mulero et al., 1998).This explanation could respond to the results foundin this study, as an excess of this type of prophylacticcompound could interfere in the optimal or posteriordevelopment of the larval immune systems (Bricknell

& Dalmo, 2005).The age at which the G MOS is applied also

affects the farming ofP. adspersus larvae. The appli-cation of G MOS at the beginning of the rst fee-ding (live prey) stage increases survival and growthas compared with the treatments given after tendays of feeding on live prey. This result challengesthe expected results, that is that older P. adspersuslarvae should have greater life expectancies (Silva,2000, 2001). Some as yet unidentied compoundsof maternal origins might provide innate protectionagainst opportunistic bacteria. Eggs are rich in somenon-specic defense compounds such as lectins andlysosomes (that probably come from the ovary) thatdifferentiate macrophages early in the embryonicdevelopment (Ellis, 2001). Nevertheless, thesecompounds decrease with larval age (Browman etal., 2003). Thus, the application of G MOS shouldcomplement and strengthen the immunity of the P.adspersus larvae that is transmitted by the motherduring the early stages of development, mainly

after the period in which the larvae have a yolk sac.However, this innate defense mechanism of the eggsand larvae seems to depend on the mothers state ofhealth (Ellis, 2001; Browman et al., 2003).

The temporal increase in the standard length ofthe P. adspersus larvae in the experimental groupspresented a similar tendency as that observed in priorstudies (Silva & Flores, 1989; Silva 2000, 2001).However, when compared with these studies, thelarvae treated with G MOS had higher growth rates,particularly when treated with 5 mgL-1 G MOS.Nevertheless, this result cannot be completely attri-buted to the effect of the G MOS application sincethese differences could be related to extrinsic factorssuch as temperature, light, and other environmentalfactors (Silva & Flores, 1989; Downing & Litvak,1999; Silva 2000, 2001; Skjermo & Bergh, 2004) thatcould have a synergic effect with the concentrationof G MOS applied to the rearing water.

In our experiment, the tissues of the intestinalepithelium revealed cells with circular, well-dened,mid-sized nuclei; these characteristics, according tothe literature, dene the monocytes (Esteban et al.,1994). The monocytes that are macrophage precur-sors are key cells in the non-specic defense systemof the sh (Dalmo et al., 1997). Such cells may haveone or several nuclei and are able to endocyte and tophagocyte foreign bodies that enter to the organism(1-10 m in size) due to the presence of glycoproteinreceptors in their plasmatic membrane that modify

their means for secretions and marker substances(Esteban et al., 1994; Dalmo et al., 1997). When themacrophages are stimulated, they produce an inter-mediate reaction of oxygen, nitric oxides, enzymes,lysozymes, cytokines, and other marker molecules(Bricknell & Dalmo, 2005). The high frequency ofmonocytes in the intestinal tissues and the greatergrowth and survival of the individuals treated withG MOS suggest a positive effect on the health ofthe P. adspersus larvae.

For P. adspersus individuals living in captivity,vibriosis is an opportunistic pathological processassociated with situations of stress such as changesin the diet and/or increased temperature; it can betreated with wide-spectrum antimicrobial agents(Miranda & Rojas, 1993, 1996). Further experimentswith this type of opportunistic pathogenic strain andP. adspersus larvae previously treated with G MOSbaths could complement our results, also consideringthe intrinsic variability of the responses of individualsfrom a single cohort.

This study is the rst to indicate that the applica-tion of G MOS (5 mgL-1 G MOS) diluted in therearing water for ve days signicantly increases thepercentage of survival as compared with the control


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situation, or as compared to other previous studiescarried out with P. adspersus larvae (Silva & Flores,

1989; Silva, 2000, 2001). The degree of protectionobtained by administering this compound is probablyrelated to the stimulation of the non-specic compo-nents of the immune system that have antibacterialactivity, as shown by the presence of macrophageprecursor cells. Other studies carried out on at sh(e.g., Hippoglossus hippoglossus, Scophthalmus

maximus, Paralichthys olivaceus) have suggestedthat the administration of compounds containing-glucans improves resistance against opportunisticbacteria (Robertsen et al., 1994; Dehasque et al.,1997; Strand & Dalmo, 1997; Sakai, 1999; Dalmoet al., 1997, 2000; Bergh et al., 2001; Bricknell &Dalmo, 2005). It is in this context that G MOS, orother products acting as prophylactic compounds,should be considered as a tools for prevention whenplanning and developing management schemes forthe intensive production ofP. adspersus juveniles,particularly during the rst feeding (live prey) stageof the larvae.

ACKNOWLEDGEMENTS

The authors would like to thank the Laboratorio deCultivo Peces of the Facultad de Ciencias del Mar,Universidad Catlica del Norte, for providing theinfrastructure for carrying out the present research.They are also grateful for the disposition of theMasters candidate Marcia Oliva and the team thatmakes up the Unidad de Produccin of the Facultadde Ciencias del Mar. They further thank Mr. AlexisRuiz (DESPRO S.A.) for providing the compoundused in the experiments. Finally, the authors appre-ciate the insightful comments and suggestions made

by the anonymous reviewers that were helpful inelaborating the nal version of this study.

REFERENCES

Anderson, D. 1992. Immunostimulants, adjuvants andvaccine carriers in sh: application to aquaculture.Ann. Rev. Fish Dis., 2: 281-307.

Angeles, B. & J. Mendo. 2005. Crecimiento, fecun-didad y diferenciacin sexual del lenguado Parali-

chthys adspersus (Steindachner) de la costa centraldel Per. Ecol. Apl., 4: 105-112.

Bergh, ., F. Nilsen & O.B. Samuelsen. 2001.Diseases, prophylaxis and treatment of the Atlantic

halibut Hippoglossus hippoglossus: a review. Dis.Aquat. Org., 48: 57-74.

Bisbal, G. & D. Bengtson. 1995. Effects of delayedfeeding on survival and growth of summer ounderParalichthys dentatus larvae. Mar. Ecol. Prog. Ser.,121: 301-306.

Bricknell, I. & R.A. Dalmo. 2005. The use of immuno-stimulants in sh larval aquaculture. Fish ShellshImmunol., 19: 457-472.

Browman, H., J.F. St-Pierre, A.B. Skiftesvik & R.G.Racca. 2003. Behaviour of Atlantic cod (Godusmorhua) larvae: an attempt to link maternal con-

dition with larval quality. In: J. Browman & A.Skiftesvik (ed.). The Big Fish Bang Proceedingsof the 26th Annual Larval Fish Conference. Bergen,Norway, pp. 72-94.

Cousin, J.C.B., G. Balouet & F. Baudin-Laurencin.1986. Alteration histoligiques observes chez deslarves de turbot (Scophthalmus maximus L) en l-vage intensive. Aquaculture, 52: 173-189.

Dalmo, R.A., K. Ingebrigtsen & J. Bgwald. 1997.Non-specic defence mechanisms in sh, with par-ticular reference to the reticuloendothelial system(RES). J. Fish Dis., 20: 241-273.

Dalmo, R., A. Kjerstad, S. Arnesen, P. Tobias & J.Bgwald. 2000. Bath exposure of Atlantic halibut(Hippoglossus hippoglossus) yolk sac larvae to

bacterial lipopolysaccharide: absorption and distri-bution of the LPS and effect on sh survival. FishShellsh Immunol., 10: 107-128.

Dehasque, M., J. van Assche & B. Deberse. 1997.Evaluacin de los efectos de la administracinoral de inmunoestimulantes en las enfermedadesde especies para acuicultura. [http://w3.dsi.uanl.mx/publicaciones/maricultura/acuicolaIII/pdfs/6.pdf], Revised: 10 June 2006

Downing, G. & M.K. Litvak. 1999. The effect of pho-toperiod, tank colour and light intensity on growthof larval haddock. Aquacult. Int., 7: 369-382.

Ellis E. A. 2001. Innate host defence mechanism of shagainst viruses and bacteria. Dev. Comp. Immunol.,25: 827-839.

Esteban, M.A., J. Muoz, A. Lpez-Ruiz, V. Mulero& J. Meseguer. 1994. Respuesta inespecficaen peces frente a las infecciones bacterianas. In-amacin, fagocitosis. In: S. Zamora, B. Aguilleiro& M.P. Garca-Hernndez (eds.). Aulas del Mar.Universidad de Murcia, Murcia, pp. 235-244.

Kumari, J. & P.K. Sahoo. 2006. Dietary -1,3 glucanpotentoes innate immunity and disease resistance ofAsian catsh, Clarias batrachus (L.). J. Fish Dis.,29: 95-101.


9/9


Kuronuma, K. & K. Fukusho. 1984. Rearing ofmarine sh larvae in Japan. Otawa, Ont. IDRC,190 pp.

Luizi, F.S., B. Gara, R. Shield & N. Bromage. 1999. Further description of the development of the di-gestive organs in Atlantic halibut (Hippoglossushippoglossus) larvae, with notes on differentialabsorption of copepod andArtemia prey. Aquacul-ture, 175: 101-116.

Miranda, C.D. & R. Rojas. 1993. Prevalencia de pa-tologas oportunistas en el cultivo experimental dellenguado Paralichthys adspersus. Anal. Microbiol.,1: 51-54.

Miranda, C.D. & R. Rojas. 1996. Vibriosis en el len-guado Paralichthys adspersus (Steindachner 1867)en cautiverio. Rev. Biol. Mar., 31(1): 1-9.

Muetn-Gmez, M. del S., M. Villarejo-Fuerte& G. Garca-Melgar. 2000. Manual de tcnicashistolgicas aplicadas a organismos marinos.Universidad Autnoma de Baja California del Sur.Publicacin del Centro Interdisciplinario de Cien-cias Marinas (CICIMAR), 81 pp.

Mulero, V., M. Esteban, J. Muoz & J. Meseguer.1998. Dietary intake of levamisole enhances theimmune response and disease resistance of marineteleost gilthead seabream (Sparus aurata L.). FishShellsh Immunol., 8: 49-62.

Pryor, G., B. Royes, F. Chapman & R. Miles. 2003.Mannanooligosaccharides in Fish nutrition: effectsof dietary supplementation on growth an gastroin-testinal villi structure in Gulf of Mexico Sturgeon.N. Am. J. Aquacult., 65: 106-111.

Raa, J. 2000. The use of immune-stimulants in shand shellsh feeds. [http://w3.dsi.uanl.mx/publica-ciones/maricultura/acuiculturaV/raa.pdf]. Revised:3 January 2006.

Ribeiro, L., C. Sarasquete, & M.T. Dinis. 1999.Histological and histochemical development of thedigestive system ofSolea senegalensis (Kaup, 1858)larvae. Aquaculture, 171: 293-308.

Robertsen, B., R.E. Engstad & J.B. Jorgensen. 1994.-Glucans as immunostimulants. Modulators of shimmune responses. In: J. Stolen & T.C. Fletcher.Fair Haven, SOS., 1: 83-99.

Rottmann, R.W., R. Francis-Floyed & R. Durborow.

1992. The role of stress in sh disease. Publication No.474, Southern Regional Aquaculture Center, 3 pp.

Sakai, M. 1999. Current research status of sh immu-nostimulants. Aquaculture, 172: 63-92.

Silva, A. 2000. Bases biolgico-tcnicas para el desar-

rollo del cultivo articial del lenguado chileno (g-nero Paralichthys). Tesis de Doctorado en CienciasBiolgicas. Universidad de Barcelona. Barcelona,122 pp.

Silva, A. 2001. Advances in the culture research ofsmall-eye flounder, Paralichthys microps, andChilean ounder, P. adspersus, in Chile. J. Appl.Aquacult., 11: 147-164.

Silva, A. & H. Flores. 1989. Consideraciones sobreel desarrollo y crecimiento larval del lenguado(Paralichthys adspersus, Steinadchner, 1987) cul-tivado en laboratorio. Rev. Pacco Sur, NmeroEspecial: 629-634.

Silva, A. & F. Castell. 2005. Tcnicas de produccinde huevos y larvas de peces. In: A. Silva (ed.).Cultivo de peces marinos. Universidad Catlica del

Norte, Coquimbo, pp. 159-184.Silva, A. & A. Vlez. 2005. Cultivo de alimento vivo

para larvas de peces marinos In: A. Silva (ed.).Cultivo de peces marinos. Universidad Catlica delNorte, Coquimbo, pp. 61-100.

Skjermo, J. & O. Vadstein. 1999. Techniques formicrobial control in the intensive rearing of marinelarvae. Aquaculture, 177: 333-343.

Skjermo, J. & . Bergh. 2004. High-M alginate im-munostimulation of Atlantic halibut (Hippoglossushippoglossus L.) larvae usingArtemia for delivery,increases resistance against vibriosis. Aquaculture,238: 107-113.

Sokal, R.R. & F.J. Rohlf. 1981. Biometry: principlesand practice of statistical in biological research. W.H.Freeman & Company, San Francisco, 776 pp.

Strand, H. & R. Dalmo. 1997. Absorption of immu-nomodulating ((1, 3)-D-glucan) in yolk sac larvaeof Atlantic halibut,Hippoglossus hippoglossus (L.).J. Fish Dis., 20: 41-49.

Servicio Nacional de Pesca (SERNAPESCA). 2005.

Diagnstico ambiental de la acuicultura chilena enfuncin de los estndares establecidos en el Regla-mento Ambiental para la Acuicultura (RAMA). De-partamento de Administracin Pesquera, 44 pp.

Tytler, P. & J.H. Blaxter. 1988. The effects of externalsalinity on the drinking rates of the larvae of herring,plaice and cod. J. Exp. Biol., 138: 1-15.

Vadstein, O. 1997. The use of immunostimulation inmarine larviculture: possibilities and challenges.Aquaculture, 155: 401-417.

Jin, Z. & L. Xiao-ling. 2004. The use of peptidolglycan

as an immune stimulant for turbot (Scophthalmusmaximus). J. Fish Soc. Taiwan, 31: 155-158.

Received: 23 October 2006; Accepted: 31 July 2007

13. c6. betaglucan

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