Aquaculture research, tập 42, số 5, 2011

REVIEW ARTICLEParticularities of early life stages in temperate freshwater fish species: comparisons with marine species and implications for aquaculture practices Fabrice Telehea & Pasc

This special issue presents a selection of the

experi-ence papers presented as posters at ‘‘larvi 2009’’, held

at Ghent University, Belgium, September 7^10, 2009

The ‘‘larvi’’ symposia (1991, 1995, 2001, 2005 and

2009) are among the few international scienti¢c

symposia, which are completely dedicated to larval

¢sh and shell¢sh research

‘‘larvi 2009’’ was co-organised by the UGent

Aqua-culture R&D consortium of Ghent University

(Bel-gium), the Norwegian University of Science and

Technology,Trondheim (Norway) and the COST action

Larvanet.‘‘larvi 2009’’was organised under the

patron-age of His Majesty Albert II, King of Belgium and was

sponsored in part by the Flemish Interuniversity

Coun-cil, the Research Council of Norway, the Norwegian

University of Science and Technology, the Province of

East Flanders and the Flemish Science Foundation

We would like to thank the members of the poster

selection committee Karin Pittman, Sadasivam

Kaushik, Ronaldo Cavalli, Ivar Ronnestad, Elin vik, Jose¤ Zambonino, Giorgos Koumoundouros, GreteBaeverfjord, Kristin Hamre, Amos Tandler, GordonBell, Bill Koven, Patrick Kestemont, Luis Conceicao,Kangsen Mai, Manuel Yufera, Atsushi Hagiwara,Yngvar Olsen, Clara Boglione, Dominique Adriaens,Maria Theresa Dinis, Lewis Le Vay, Konrad Dabrows-

Kjrs-ki, Trine Galloway, Peter Bossier, Olav Vadstein andPavlos Makridis for their thorough work in reviewingall the poster contributions

The review papers presented at larvi 2009 are lished in Aquaculture (in press) Pdf ¢les of most ofthe oral and poster presentations can be found atwww.larvi.ugent.be

pub-Patrick Sorgeloos,larvi 2009 conference chairman

REVIEW ARTICLE

Particularities of early life stages in temperate

freshwater fish species: comparisons with marine species and implications for aquaculture practices

Fabrice Telehea & Pascal Fontaine

URAFPA, Nancy Universite¤ ^ INRA,Vandoeuvre-le's-Nancy, France

Correspondence: F Teletchea, URAFPA, Nancy-Universite¤ ^ INRA, 2 avenue de la ForeŒt de Haye, F-545000 Vandoeuvre-le's-Nancy, France E-mail: fabrice.teletchea@lsa-man.uhp-nancy.fr

Abstract

Both egg and larvae are di¡erent between freshwater

and marine ¢sh species Freshwater ¢sh species have

generally larger and fewer eggs than marine species

Most freshwater ¢sh species have demersal eggs that

develop stuck to various substrata, such as plants or

gravels, while eggs of most marine ¢sh species

devel-op in the water column These di¡erences have

con-sequences for both the evaluation of the quality and

the incubation of eggs of freshwater ¢sh species

com-pared with marine species The larvae of many

fresh-water ¢sh species are larger and more developed at

hatching than their marine counterparts: thus,

lar-val feeding regimes could be di¡erent and

cannibal-ism may emerge sooner in certain freshwater ¢sh

species The main di¡erences of egg and larvae

be-tween freshwater and marine species are highlighted

and the possible implications for aquaculture

prac-tices are discussed

Keywords: domestication, egg, larvae, marine,

freshwater

Introduction

In the past 50 years, aquaculture production has

grown at an average annual rate of nearly 7 per cent,

starting from a production ofo1 million tonnes per

year in the early 1950s to 51.7 million tonnes in 2006

(FAO 2009) Considered to be the fastest growing

glo-bal primary industry, aquaculture is for the ¢rst time

set to produce half of the ¢sh consumed by thehuman population worldwide, and is expected tomaintain an average annual growth rate of 44%over the period 2010^2030 (Bruge're & Ridler 2004;FAO 2009) This re£ects not only the vitality of theaquaculture sector but also global economic growthand continuing developments in ¢sh processing andtrade (FAO 2009) Yet, such a rapid development hasbeen questioned on environmental grounds, in parti-cular its dependence on ¢shmeal supplies for aquaticfeeds, which in turn depend on approximately 25% ofthe dwindling marine capture ¢shery (Naylor, Gold-burg, Primavera, Kautsky, Beveridge, Clay, Folke, Lub-chenco, Mooney & Troell 2000; Tacon & Metian2008) in conjunction with its potential impacts onbiodiversity, chie£y due to the introduction of alienspecies (Hall & Mills 2000; Manchester & Bullock2000; Casal 2006; De Silva, Nguyen, Abery & Amar-asinghe 2006; Innal & Erk’akan 2006; De Silva,Nguyen, Turchini, Amarasinghe & Abery 2009; Dia-

na 2009) In this relatively young food production dustry, mitigating the dependence on alien specieswhile promoting local production of indigenous spe-cies, in accordance with regional consumer demandand proximity to consumption areas, is imperativefor a sustainable future (Lee 2003; Muir 2005; De Sil-

in-va et al 2009; Fontaine, Legendre, Vandeputte & tier 2009)

Fos-In 2007, world ¢n¢sh production reached nearly

31 million tonnes, which came primarily from water species (28 million tonnes), followed by dia-dromous species (2 million tonnes) and marine

species (1 million tonnes) (http://www.fao.org);

in-terestingly, 85% of this total came from 15 species

alone (Lazard & LeveŒque 2009) In Europe, ¢n¢sh

production was approximately 1.6 million tonnes in

2007 (http://www.fao.org) This total output was

dominated by the marine production of three

spe-cies, these being Atlantic salmon (Salmo salar),

European seabass (Dicentrarchus labrax) and gilthead

seabream (Sparus aurata) (Lee 2003; Suquet,

Diva-nach, Hussenot, Coves & Fauvel 2009) However,

fol-lowing recent targeted e¡orts to diversify the

European marine production, there were also small

contributions from other species such as Atlantic

cod (Gadus morhua), Atlantic halibut (Hippoglossus

hippoglossus), meagre (Argyrosomus regius) and sole

species (Solea spp.) (Suquet et al 2009) Concerning

inland production, 65% of the total volume was

based on alien species, among which the most

im-portant were rainbow trout (Oncorhynchus mykiss),

silver carp (Hypophthalmichthys molitrix) and

com-mon carp (Cyprinus carpio) (Turchini & De Silva

2008) As with the marine sector, a few other

species, including Eurasian perch (Perca £uviatilis),

pikeperch (Sander lucioperca), burbot (Lota lota) and

tench (Tinca tinca), are considered as potential

candi-dates for the diversi¢cation of inland production in

relation to either large or local market demands

(Fontaine 2009)

The ¢sh life cycle is commonly divided into ¢ve

periods: embryo, larvae, juvenile, adult and

senes-cence, despite the ‘decisive’ threshold separating each

period being open to debate (Balon 1984; KovaŁc› &

Copp 1999; Pen›aŁz 2001; Kamler 2002; Urho 2002)

The sustainable production of a new species requires

gathering biological and zootechnical knowledge on

these ¢ve periods (Falk-Petersen 2005; Bilio 2008;

Bobe & Labbe¤ 2010) This includes, among other

con-siderations, the environmental control of the

repro-duction of breeders, the incubation of eggs and the

subsequent rearing of larvae and juveniles In

re-sponse to a previous extensive analysis of the

litera-ture by Teletchea, Fostier, Le Bail, Jalabert, Gardeur

and Fontaine (2007), the three main goals of the

pre-sent study were (i) to provide an updated review of

the knowledge acquired about the early life stages

(eggs and larvae) of freshwater temperate ¢sh

cies, (ii) to show their di¡erences with marine

spe-cies and (iii) to highlight the implications for

aquaculture practices This review is part of a wider

project aimed at developing a general approach to

promote the domestication of new ¢sh species,

parti-cularly those inhabiting European waters (Teletchea,

Fostier, Kamler, Gardeur, Le Bail, Jalabert & Fontaine2009)

EggFreshwater ¢sh species generally have fewer but lar-ger eggs than marine species, a di¡erence that is notdirectly attributable to di¡erences in body size be-tween freshwater and marine ¢sh species (Elgar1990) However, both freshwater and marine speciesshow a signi¢cant positively skewed distribution ofegg diameters (Kamler 2005; Teletchea, Gardeur,Kamler & Fontaine 2009) Most freshwater ¢sh spe-cies produce demersal eggs that adhere to varioussubstrata, such as plants or gravels where they devel-op; while the same is true for some marine species(Lnning, Kjrsvik & Falk-Petersen 1988), the major-ity of eggs are pelagic (Ware1975; Houde1994; Hirst &Lopez-Urrutia 2006; Teletchea, Gardeur et al 2009).These di¡erences may have consequences for boththe evaluation of the quality and the incubation ofeggs of freshwater ¢sh species compared with marinespecies, as discussed further below

Egg qualityEgg quality can be de¢ned as the ability of the egg to

be fertilized and subsequently develop into a normalembryo (Kjrsvik, Mangor-Jensen & Holmefjord1990; Bobe & Labbe¤ 2010) Despite extensive re-search, variable egg quality remains one of the mainlimiting factors for the successful mass production of

¢sh larvae for both freshwater and marine ¢sh cies (Kjrsvik et al 1990; Kamler 2005; Bobe & Labbe¤2010) Some of the key factors a¡ecting egg qualityinclude maternal attributes (age, size, fecundity),broodstock feeding and the environmental condi-tions (photoperiod, temperature, stress) under whichthe broodstock are reared and the physico-chemicalparameters of the water (temperature, salinity, oxy-gen, pH) in which the eggs are incubated, but manyare still unknown (Dabrowski 1984a; Kjrsvik et al.1990; Tyler & Sumpter 1996; Brooks,Tyler & Sumpter1997;Thorsen,Trippel & Lambert 2003; Kamler 2005;Bobe & Labbe¤ 2010) Recent studies focusing on therole of some maternal mRNAs have also providedsome hints on the molecular mechanisms involved

spe-in the regulation of egg quality spe-in ¢sh (reviewed spe-inBobe & Labbe¤ 2010; Lubzens, Young, Bobe & CerdaØ2010) The identi¢cation of predictive estimators ormarkers of egg quality would have major applicationsAquaculture Research, 2011, 42, 630^654 Early life-stages in freshwater ¢sh F Teletchea & P Fontaine

in aquaculture (Bobe & Labbe¤ 2010); however, to date,

no such estimators or markers have been found

Current methodologies allow non-viable gametes to

be identi¢ed in select species through the assessment

of simple parameters such as buoyancy or

appear-ance (Kjrsvik et al 1990; Lahnsteiner, Urbanyi,

Hor-vath & Weismann 2001; Thorsen et al 2003; Bobe &

Labbe¤ 2010) For example, in marine species with

buoyant eggs, such as Atlantic cod, unfertilized eggs

sink to the bottom of the tank, while fertilized eggs

£oat (Thorsen et al 2003; Sawanboonchun, Roy,

Ro-bertson & Bell 2008) For some freshwater species,

such as Eurasian perch or Arctic charr (Salvelinus

al-pinus), fertilized eggs have a translucent appearance

while unfertilized eggs have a whitish appearance or

are opaque (Huuskonen, Penttinen & Piironen 2003;

Migaud, Wang, Gardeur & Fontaine 2004) Therefore

currently, the only biologically relevant ways

avail-able to consistently assess egg quality for either

fresh-water or marine ¢sh species are fertilization and

hatching rates, survival to speci¢c developmental

stages, larval malformations (scoliosis, lordosis),

mal-pigmentation or larval stress tests (Kjrsvik et al

1990; Dhert, Lavens & Sorgeloos 1992; Abi-Ayad,

Me¤lard & Kestemont 1997; Planas & Cunha 1999;

Emata, Borlongan & Damaso 2000; Kjrsvik,

Hoehne-Reitan & Reitan 2003; Thorsen et al 2003;

AŁlvarez, Racotta, Arjona & Palacios 2004; Avery,

Killen & Hollinger 2009; Bobe & Labbe¤ 2010)

Implications for egg incubation

The physico-chemical parameters related to the

water (temperature, salinity, oxygen, light intensity,

pH, xenobiotic) in which eggs are incubated are key

factors in£uencing their quality (Alderdice 1985;

Brooks et al 1997; Kamler 2002) Among these

di¡er-ent physico-chemical parameters, water temperature

is the most important for both freshwater and marine

species, followed by salinity for marine species

(Mill-er, Crowd(Mill-er, Rice & Marschall 1988; Blaxter 1992;

Brooks et al 1997; Kamler 2002; Teletchea, Gardeur

et al 2009) Indeed, the temperature at which eggs

are incubated can a¡ect not only their quality but

also the tissue di¡erentiation rate, the activity of

hatching glands and embryo motility (Elliott,

Hum-pesch & Hurley 1987; Pepin 1991; Brooks et al 1997;

Kamler 2002) Over 90% of the variation in the

em-bryo ontogenetic rate is controlled by temperature

(Kamler 2002) At the intraspeci¢c level, it is now

well established that within a viable temperature

range, the time required by fertilized ¢sh eggs to cubate decreases with increasing temperature, withall other factors being equal This negative correla-tion has been found for both marine ¢sh species,e.g for Atlantic cod (Ge¡en, Fox & Nash 2006) or had-dock (Melanogrammus aegle¢nus) (Martell, Kie¡er &Trippel 2006) and freshwater ¢sh species, such assalmonids (Elliott et al 1987) or cyprinids (Keckeis,Kamler, Bauer-Nemeschkal & Schneeweiss 2001;Kupren, Mamcarz, Kucharczyk, Prusinska, Krejsze¡2008) At the interspeci¢c level, a negative relation-ship between incubation time and water temperaturewas also found for marine ¢sh species (Pauly & Pullin1988; Pepin 1991) and freshwater ¢sh species (Tele-tchea, Gardeur et al 2009) (Table1) Egg diameter alsoslightly in£uences the incubation time in both mar-ine and freshwater ¢sh species (Pauly & Pullin 1988;Pepin 1991; Bonislawska, Formicki & Winnicki 2000;Teletchea, Gardeur et al 2009) However, equationsbased on marine species (Pauly & Pullin 1988; Pepin1991) poorly ¢t the dataset of freshwater species (Tel-etchea, Gardeur et al 2009) primarily because themodel greatly underestimates incubation time, espe-cially for the lowest temperatures (see Teletchea, Gar-deur et al 2009) Consequently, the equationsobtained from marine species to predict the incuba-tion time based on either water temperature and/oregg diameter cannot be applied to freshwater ¢shspecies and vice versa (Table 1) More generally, ithas been demonstrated that egg diameter alone can-not accurately predict the incubation time This is be-cause ¢rstly, it only partly corresponds to the amount

in-of reserves (yolk) and secondly, the caloric values in-ofegg dry matter varies considerably between species(Balon 1986; Lnning et al 1988; Wiegand 1996; Boni-slawska, Formicki, Korzelecka-Orkisz & Winnicki2001; Kamler 2005; Teletchea & Fontaine 2010).Eggs of teleost ¢sh are surrounded with a relativelythick proteinaceous layer, which is called the chor-ion, egg shell or zona radiata (Lnning et al 1988;Kunz 2004; Lubzens et al 2010) The zona radiata hasboth structural and morphological di¡erences de-pending on the systematic position and ecology of

¢sh species (Riehl & Patzner 1998; Kunz 2004) Thisenvelope normally consists of two layers, a zona radia-

ta interna and a zona radiata externa (Riehl & Patzner1998; Mansour, Lahnsteiner & Patzner 2009) Innumerous freshwater ¢sh species and particularlycyprinids, the zona radiata externa becomes sticky

in contact with water, thus enabling eggs to attach

to each other and to aquatic substrata, such as plants

or gravels (Riehl & Patzner 1998; Mansour et al 2009;Early life-stages in freshwater ¢sh F Teletchea & P Fontaine Aquaculture Research, 2011, 42, 630^654

Teletchea, Fostier et al 2009) In cyprinids, arti¢cial

incubation is usually carried out in inverted bottles

provided with a continuous water £ow (Carral,

Cela-da, SaŁez-Royuela, Rodr|¤guez, Aguilera & Melendre

2006) In these systems, the reduction in egg

sticki-ness is recommended in order to assure the success

of the incubation (Gela, Linhart, Flajshans & Rodina

2003; Carral et al 2006) Many methods have been

developed for removing the stickiness of ¢sh eggs:

se-parating individual eggs mechanically, scouring

them physically with abrasives (¢ne clay and/or talc

suspensions) or treating them chemically with milk,

salt, tannic acid or enzymes (Table 2) Gela et al

(2003) compared four methods to reduce the egg

stickiness in tench: alcalase enzyme, milk powder

with talc suspension, ¢ne clay suspension and talc

suspension They found that each procedure was

suc-cessful, with neither the destruction of egg envelopes

nor larval malformations being observed They also

found that the alcalase technique increased the

hatching rate and required less time than the

tradi-tional milk/clay/talc treatments (Gela et al 2003)

For certain cyprinid species, e.g barbel (Barbus

bar-bus), the eggs are only slightly sticky and it is not

ne-cessary to apply any particular method for removing

the stickiness before incubation (Krupka 1988;

Krup-ka & Meszaros 1993)

In conclusion, the eggs of freshwater ¢sh species are

generally di¡erent from those of marine

species.With-in freshwater species, eggs are very diverse species.With-in their

dia-meter, buoyancy, stickiness, incubation time or water

temperature requirement (Teletchea, Fostier et al.2009; Teletchea, Gardeur et al 2009; Teletchea &Fontaine 2010) Di¡erent types of incubators have beendeveloped according to the speci¢city of the eggs of thetargeted freshwater ¢sh species (Table 3)

LarvaeMorphological development and larval size

at hatchingHatching is usually considered to be the beginning ofthe larval period, despite some authors consideringthat the larval period begins either at the moment ofthe onset of exogenous feeding or after the completeresorption of the yolk sac (for further discussion onthis, see Pen›aŁz 1983; Balon 1984, 1986; Hensel 1999;KovaŁc› & Copp 1999; Pen›aŁz 2001; Kamler 2002, 2008;Urho 2002) Nevertheless, hatching is a major turn-ing point from ecological, physiological and beha-vioural points of view (Pen›aŁz 2001; Kamler 2002) Asubstantial variability in the stage of morphologicaldevelopment at hatching was found both betweenand within marine and freshwater ¢sh species (Pe-n›aŁz 1983, 2001; Miller et al 1988; Falk-Petersen 2005;Teletchea & Fontaine 2010) For instance, Lnning

et al (1988) found that the larvae from halibut (H poglossus) hatch at a very premature stage comparedwith the larvae from lumpsucker (Cyclopterus lum-pus), which hatch at a more advanced stage Whencomparing 17 taxa of ¢sh belonging to the Salmonoi-

hip-Table 1 Relationships between oocyte diameter ( +), incubation time (t), water temperature (T) and larval size at hatching (L) for teleost, mostly temperate, ¢sh species

Variables Species Equations r 2

n

Range of + (mm)

Aquaculture Research, 2011, 42, 630^654 Early life-stages in freshwater ¢sh F Teletchea & P Fontaine

dei, Pen›aŁz (1983) found that hatching was

species-speci¢c, occurring between the seventh and the 11th

developmental steps (on a developmental scale with

12 steps), and depended mainly on egg size and

vo-lume of the yolk Teletchea and Fontaine (2010)

de-monstrated that the developmental stages at

hatching among 65 freshwater temperate ¢sh species

were not ¢xed in ontogeny and were not directly

re-lated to either larval size or degree-days for

incuba-tion, but were probably species-speci¢c This implies

that morphological and physiological development

proceeds much further inside the egg shell in somespecies than in others (Balon 1986; Hensel 1999;Urho 2002) At the intraspeci¢c level, a substantialvariability in the developmental stage at hatchingwas also observed depending on physico-chemicalfactors, such as temperature and dissolved oxygen le-vels (Pen›aŁz 1983; Blaxter 1992; Urho 2002; Jordaan,Hayhurst & Kling 2006) Conspeci¢c larvae hatchingfrom eggs incubated at higher temperatures are gen-erally shorter in total body length (Blaxter 1992;Jordaan et al 2006) Moreover, within a single egg

Table 2 Main products tested for removing the stickiness of ¢sh eggs

Species Milk Clay Talc NaCl Urea

Tannic acid Kaolin Alcalase Trypsin Protease References

Cyprinidae

Chondrostoma

nasus

(1981)

Watchorn, Kollar and Casselman (2007) Siluridae

Silurus

glanis

Grecu and Billard (2002)

Product can be either used alone or mix in the same solution.

Early life-stages in freshwater ¢sh F Teletchea & P Fontaine Aquaculture Research, 2011, 42, 630^654

Table 3 Examples of devices used for incubating temperate freshwater ¢sh eggs

Species

Zoug jar Weiss jar Tray-type incubators

Prunet and Bagliniere (2007)

egg incubation jars

Wiggins, Bender, Mudrak and Coll (1985)

Cobitidae

Cyprinidae

Targonska-Dietrich, Wyszomirska, Glogowski and Szabo (2005)

Glass plates put in containers

Gerasimov and Stolbunov (2007)

chambers

Winnicki and Korzelecka (1997)

(2007)

Plastic Chase type flasks Halacka and Lusk (1995) Nylon screens in plastic

boxes

Keckeis, Bauer-Nemeschkal, Menshutkin, Nemeschkal and Kamler (2000)

Hypophthalmichthys

molitrix

Poluektova (2006)

Mamcarz, Kujawa, Kaszkowski and Ratajski (2008)

McAllister, Beresford, Henshaw, Brighty, Tyler and Sumpter (2002)

Wamuini and Philippart (2007)

Stibranyiova and Asenjo (1995)

Aquaculture Research, 2011, 42, 630^654 Early life-stages in freshwater ¢sh F Teletchea & P Fontaine

Table 3 Continued

Species

Zoug jar Weiss jar Tray-type incubators

Petri

Esocidae

Flow-through hatchery cones

Vehnia¨inen, Ha¨kkinen and Oikari (2007)

Moronidae

Ictaluridae

Crawford (2004) Percidae

tank

Jentoft, Held, Malison and Barry (2002)

and Dixon (2007)

apparatus

Korzelecka, Bonislawska and Winnicki (1998)

(2007)

Salmonidae

Coregonus

clupeaformmis

Hatchery jars with continuously upwelling water

Drouin, Kidd and Hynes (1986)

Coregonus

lavaterus

Floating small boxes

in tank

Olsen and Vollestad (2001)

Early life-stages in freshwater ¢sh F Teletchea & P Fontaine Aquaculture Research, 2011, 42, 630^654

batch from the same parents that experiences a

com-mon environment during its development, hatching

is not completely synchronous and, especially at

low-er templow-eratures, sevlow-eral days can elapse between the

earliest and the latest hatching larvae (Ge¡en 2002;

Jordaan et al 2006; Laurel, Hurst, Copeman & Davis

2008) Larvae hatching at the beginning of the

hatch-ing period are generally shorter, with larger yolk

sacs, than individuals hatching later in the hatching

period (Ge¡en 2002; Jordaan et al 2006; Laurel et al

2008) Like hatching, the onset of exogenous feeding

and the full resorption of the yolk sac have enormous

physiological, ecological and behavioural

signi¢-cance and occur over a wide range of developmental

stages (Dabrowski 1984a; Blaxter 1992; Urho 2002;

Kunz 2004; Falk-Petersen 2005; Yu¤fera & Darias

2007) Yet, the stage of morphological development

during the transition from endogenous to exogenous

feeding is not accompanied by such extensive

varia-bility as is observed at hatching This is primarily

be-cause all structures and organs related to food intake,

digestion and assimilation have to be ready to ensure

this transition successfully (Pen›aŁz 1983; Miller et al

1988; Cahu & Zambonino-Infante 2001; Yu¤fera &

Darias 2007) In conclusion, there exists a wide rangeand continuous spectrum of levels of morphologicaldevelopment attained at the stage of hatching, onset

of exogenous feeding and the full absorption of theyolk sac for both marine and freshwater ¢sh species(Pen›aŁz 2001)

Larval size at hatching varies widely both withinand between freshwater and marine species (Miller

et al.1988; Pepin 1991; Chambers & Leggett tchea & Fontaine 2010) At the interspeci¢c level, asigni¢cant positive correlation was found betweenoocyte diameter and larval size at hatching amongmarine and freshwater ¢sh species (Ware1975; Miller

1996;Tele-et al 1988; Kjrsvik 1996;Tele-et al 1990; Pepin 1991; Chambers

& Leggett 1996; Teletchea & Fontaine 2010) However,the three equations based on marine species (Table 1)poorly ¢t the dataset of 65 freshwater ¢sh species(Teletchea & Fontaine 2010) primarily because themodels greatly underestimate the larval size at hatch-ing, especially for larger eggs (see Teletchea & Fon-taine 2010) This con¢rms the notion that marinespecies generally produce smaller larvae than fresh-water ¢sh species do (Balon 1984) For instance,Houde (1994) measured a 10-fold di¡erence in the

Table 3 Continued

Species

Zoug jar Weiss jar Tray-type incubators

Petri

Floating plastic cylinders in tank

Huuskonen et al (2003)

USA)

Mirza, Chivers and Godin (2001)

Salvelinus

namaycush

Linhart, Rodina, Flajshans, Gela and Kocour (2005)

Aquaculture Research, 2011, 42, 630^654 Early life-stages in freshwater ¢sh F Teletchea & P Fontaine

mean weight at hatching between marine (37.6mg;

n 5 77) and freshwater (359.7mg; n 5 20) larvae

No larva can feed before it is functionally capable

(i.e., its mouth and digestive system must have

devel-oped), but it must feed before reaching irreversible

starvation or a ‘point of no return’ (Miller et al 1988)

Miller et al (1988) and Pepin (1991) found that larger

larvae at hatching are generally more resistant to

starvation because they have more energy reserves

than small larvae, but more importantly, they have a

greater £exibility (window of opportunity) in ¢rst

feeding times The period between the mouth

open-ing and the ‘point of no return’ is species-speci¢c and

also depends on water temperature, ranging

approxi-mately from 3 days in temperate waters to 20 days in

cold waters (Yu¤fera & Darias 2007) In conclusion,

the larvae of many freshwater ¢sh species are larger

and more developed than their marine counterparts;

thus, feeding regimes could be di¡erent and

canni-balism may emerge sooner in certain freshwater ¢sh

species, as is discussed further below

Implications for the larviculture of

freshwater ¢sh species

The transition from yolk sac larvae to actively feeding

larvae is considered to be the most critical event

dur-ing the early life of larval ¢sh (Roche-Mayzaud,

May-zaud & Audet 1998; Yu¤fera & Darias 2007) To achieve

this transition successfully, all structures and organs

related with food uptake, digestion and assimilation

have to be ready in time and the appropriate food

has to be available by the time the yolk is depleted

(Yu¤fera & Darias 2007) The two main limitations at

the beginning of exogenous feeding of larvae are

mouth gape, restricting the particle size (a prey/gape

ratio of 25^50% seems to be the most appropriate),

and larval length, which restricts swimming activity

and therefore hunting success (Dabrowski 1984a;

Miller et al 1988; Planas & Cunha 1999; Yu¤fera &

Dar-ias 2007) In order to avoid mass mortalities due to

starvation, an appropriate diet must be provided to

the larvae when they ¢rst begin feeding Two main

kinds of diet are available for ¢sh larvae: live prey

and compound or formulated diet

For marine ¢sh species, the feeding regimes of

lar-vae begin with live prey, usually rotifers (Brachionus

spp.) and brine shrimp (Artemia spp.) nauplii for the

early life stages; then larvae are weaned to

formu-lated feeds (Rainuzzo, Reitan & Olsen 1997; Planas &

Cunha 1999; Langdon 2003; Lee 2003; Conceicao,

Aragao, Richard, Engrola, Gavaia, Mira & Dias 2010;Conceicao,Yu¤fera, Makridis, Morais & Dinis 2010) Inthe past 25 years, e¡orts have been made to developnew formulated diets in order to partially or totallyreplace live feeds for rearing the early larval stages ofseveral marine species (Planas & Cunha 1999; Shields2001; Langdon 2003; Lee 2003; Conceicao, Aragao

et al 2010; Conceicao,Yu¤fera et al 2010) This tion of live prey is crucial for reducing productioncosts and for sustaining the production of high- andconstant-quality juveniles (Cahu & Zambonino-In-fante 2001) Several factors must be considered whendeveloping formulated diets, including homogeneouscomposition, digestibility, nutritional value, palatabil-ity, water stability and an optimum size to ensure thatlarvae can detect and ingest them (Rainuzzo et al.1997; Planas & Cunha 1999; Cahu & Zambonino-In-fante 2001; Lee 2003; Cahu, Gisbert,Villeneuve, Mor-ais, Hamza, Wold, Zambonino-Infante 2009).Currently, formulated diets are still not adequatewhen used exclusively to rear marine ¢sh larvae(Langdon 2003; Lee 2003; Conceicao, Aragao et al.2010; Conceicao, Yu¤fera et al 2010) The poor perfor-mance of formulated diets is related partly to the in-adequate incorporation of nutrients into thediets in conjunction with poor ingestion, digestionand/or assimilation (Planas & Cunha 1999; Cahu &Zambonino-Infante 2001; Langdon 2003; Lee 2003).However, results are markedly improved whenformulated diets are co-fed with live prey (Planas &Cunha 1999; Langdon 2003) Co-feeding not only sti-mulates the ingestion of food particles but also pro-motes digestion and assimilation of formulated diets

substitu-by ¢sh larvae (Lee 2003) Any failure or limitation inthe nutrients and energy uptake during the feedingonset period a¡ects the correct development of or-gans and structures and the subsequent growth andsurvival of the larvae (Yu¤fera & Darias 2007; Shan,Huang, Cao & Wu 2008; Conceicao, Aragao et al.2010; Conceicao, Yu¤fera et al 2010) Environmentalfactors such as water temperature, salinity, light in-tensity and microalgae (green water) can all in£uencethe feeding of larvae (Dabrowski 1984a; Planas &Cunha 1999; Dou, Masuda, Tanaka & Tsukamoto2005) For instance, light is of primary importance inmarine larviculture, as most marine ¢sh larvae are vi-sual feeders (Planas & Cunha 1999; Cahu & Zamboni-no-Infante 2001; Yu¤fera & Darias 2007) Temperature

is generally considered to be a major factor in mining the advent of endogenous feeding of ¢sh lar-vae because of its direct e¡ects on their oxygenconsumption, yolk exhaustion rate, feeding activityEarly life-stages in freshwater ¢sh F Teletchea & P Fontaine Aquaculture Research, 2011, 42, 630^654

deter-and food conversion e⁄ciency (Pepin 1991; Blaxter

1992; Kamler 2002; Dou et al 2005) In intensive

rear-ing tanks in European hatcheries, the formation of an

oily ¢lm on the water surface impedes the access of

larvae to the surface and thus inhibits air intake by

physostomous larvae, particularly seabass and

seab-ream, which could lead to both skeletal deformities

and poor growth and resistance (Planas & Cunha

1999) Excessive turbulence can also inhibit in£ation

of the swimbladder (Planas & Cunha 1999)

For some freshwater ¢sh species, such as pike (Esox

lucius), coregonids (Coregonus spp.) and salmonids, it

is believed that larvae can be fed formulated diets as

early as mouth opening (Dabrowski 1984a; Cahu &

Zambonino-Infante 2001); see also Table 4 Yet, for

other species, and particularly cyprinids, the larval

ability to utilize formulated diets from the ¢rst

feed-ing is low (Dabrowski 1984a; Wolnicki 2005; Kamler

& Wolnicki 2006) Thus, feeding regimes need to

be-gin with live prey for at least 5 days, and sometimes

not less than 8^12 days are required before larvae

can be weaned to formulated feeds (Dabrowski

1984a; Wolnicki 2005) For instance, satisfactory

growth performance and/or survival were not

achieved when exclusively formulated diets were fed

to larval asp (Aspius aspius), gudgeon (Gobio gobio),

chub (Leuciscus cephalus), dace (Leuciscus leuciscus)

or tench (Wolnicki 2005; Wolnicki, Sikorska &

Kaminski 2009) In fact, only a few cyprinids

demon-strate relatively fast growth and high survival rates

when fed formulated diets exclusively from the very

beginning of exogenous feeding, these include barbel

(B barbus), gold¢sh (Carassius auratus), nase

(Chon-drostoma nasus), roach (Rutilus rutilus) and vimba

(Vimba vimba) (Wolnicki 2005; Wolnicki et al 2009)

Morphological (particularly mouth size) and

func-tional di¡erences between ¢sh species and during

ontogenesis may, to some extent, explain the

nutri-tional constraints in the acceptance and assimilation

of a formulated diet (Dabrowski1984a) To our

knowl-edge, no extensive review on the nutritional

require-ments of freshwater larvae has been performed

which matches the works on marine ¢sh species

(Pla-nas & Cunha 1999; Cahu & Zambonino-Infante 2001;

Langdon 2003; Cahu et al 2009; Conceicao, Aragao

et al 2010; Conceicao, Yu¤fera et al 2010) However, it

is clear that problems due to sub-nutrition or other

environmental parameters observed in marine ¢sh

species (such as low growth and high mortalities, as

well as malformations) are also often encountered in

the larviculture of freshwater species such as rudd

Scardinius erythrophthalmus (Wolnicki et al 2009),

Eurasian perch (Tamazouzt, Leray, Esca¡re & Terver1998; Tamazouzt, Chatain & Fontaine 2000) orpikeperch (Schlumberger, Proteau & Albiges 1993;Hamza, Mhetli, Khemis, Cahu & Kestemont 2008).Therefore, a better understanding of the speci¢c nu-tritional requirements of larvae of freshwater ¢shspecies is needed

Cannibalism is the act of killing and consumingthe whole, or a major part, of a conspeci¢c individual(Smith & Reay 1991) Cannibalistic behaviour is re-ported in a large number of both marine and fresh-water ¢sh species occupying di¡erent habitats andpursuing di¡erent life-history strategies, but it is par-ticularly common in piscivorous and parental care-giving species (Smith & Reay 1991; Hecht & Pienaar1993) Piscivores often have a large gape and well-formed teeth, allowing them to consume relativelylarge prey, which advances the onset of cannibalism(Smith & Reay 1991) Non-piscivorous species canalso exhibit sibling (intracohort) cannibalism in lar-viculture, such as common carp (Table 5) From afunctional viewpoint, ¢sh can start cannibalizing assoon as the structures required for suction feedingare developed This can take place as early as the start

of exogenous feeding in certain species, althoughmost ¢sh species start exerting cannibalism much la-ter (Baras & Jobling 2002) Owing to the fact that thelarvae of many freshwater ¢sh species are larger andmore developed at hatching than that of their marinecounterparts, cannibalism may emerge sooner(Baras & Jobling 2002) For instance, pike, pikeperchand Eurasian perch are three freshwater piscivorous

¢sh species in which cannibalism occurs quite early

in the development (Table 5) On the contrary, eventhough seabass is a piscivorous species, cannibalism

is not observed in young stages (Cahu & Infante 2001) There are two types of cannibalism:partial ingestion of prey, mainly tail ¢rst (type I), andcomplete cannibalism, with a head-¢rst ingestion ofprey (type II) (Baras & Jobling 2002; Kestemont, Jour-dan, Houbart, Me¤lard, Paspatis, Fontaine, Cuvier,Kentouri & Baras 2003) Type I precedes type II can-nibalism, because the caudal peduncle of a ¢sh

Zambonino-is generally much smaller than the gape of a ¢sh ofsimilar size Type I cannibalism does not require thepredator to prey size ratio to be large (Hecht & Pie-naar 1993; Folkvord 1997; Baras & Jobling 2002) Theswitch to type II cannibalism occurs as size heteroge-neity develops, i.e., when some ¢sh have gained su⁄-ciently from type I cannibalism to have a large sizeadvantage over others (Baras & Jobling 2002) In the-ory, intracohort type II cannibalism can persist asAquaculture Research, 2011, 42, 630^654 Early life-stages in freshwater ¢sh F Teletchea & P Fontaine

long as siblings small enough to be consumed by

the cannibal are available (Baras & Jobling 2002)

Cannibalism is thus facilitated by size heterogeneity,

but it also a¡ects size heterogeneity, as the smallest

¢sh are consumed by the largest ones, and it can be

viewed as both a cause and a consequence of size

het-erogeneity (Hecht & Pienaar 1993; Baras & Jobling

2002; Kestemont et al 2003) Even though

cannibal-ism is genetically determined (Hecht & Pienaar 1993),

many environmental biotic and abiotic factors

ap-pear to a¡ect the extent of the rate of cannibalism;

these include food availability, population density,

re-fuges, water clarity, light intensity, feeding frequency

and the frequency at which alternative prey is

pre-sented (Smith & Reay 1991; Hecht & Pienaar 1993;

Baras & Jobling 2002) In larviculture, cannibalism

can cause signi¢cant losses, particularly when the

size variation in the population is su⁄ciently large,

because of high stocking densities, lack of alternative

live food and absence of refuges from predation

(Smith & Reay 1991; Folkvord 1997; Baras & Jobling

2002) The complete elimination of cannibalism in

larviculture is probably impossible (Baras & Jobling

2002); however, it is possible to mitigate cannibalism,notably by frequent grading to reduce size variability(Smith & Reay 1991) The value of mitigating canni-balism in larviculture in both freshwater and marinespecies is a matter of cost-e¡ectiveness, which de-pends on labour costs and the productivity of rearingsystems (Baras & Jobling 2002)

The progeny of most species must disperse afterhatching (species with pelagic eggs start earlier) andothers after a short inactive period (Urho 2002) Theway in which larvae of di¡erent species disperse seems

to depend on their morphology and development stage

at hatching (Urho 2002) In freshwater ¢sh species,some remain nearly motionless during the entire per-iod of yolk resorption, such as pike or bream (Abramisbrama), while others, such as burbot or pikeperch, dis-perse immediately after hatching (Teletchea, Fostier

et al 2009;Teletchea & Fontaine 2010)

ConclusionsBoth egg and larvae di¡er in many respects betweenmarine and freshwater ¢sh species These di¡erenceshave consequences for aquaculture practices, parti-cularly for the evaluation of the quality of the egg,the incubation of adhesive eggs, the ¢rst feeding oflarvae and the onset of cannibalism Further experi-ments are required to improve the current methodsfor removing the adhesiveness of eggs of freshwater

¢sh species, particularly for some cyprinids In tion, studies that focus on the speci¢c nutritional re-quirements of larvae of freshwater ¢sh species,including the in£uence of dietary phospholipids(Cahu et al 2009) and the importance of live preyand formulated diets in larviculture (Shields 2001;Conceicao, Aragao et al 2010; Conceicao,Yu¤fera et al.2010), are needed in order to improve growth and re-duce both mortalities and deformities

addi-Acknowledgment

We thank Andrew Davie and two anonymous viewers who helped to improve the manuscript

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Dietary soybean phosphatidylcholine affects growth performance and lipolytic enzyme activity in Caspian

Abdolmohammad Abedian Kenari1, Ebrahim Sotoudeh1& Mehran Habibi Rezaei2

1 Department of Fisheries, Faculty of Natural Resources and Marine Sciences,Tarbiat Modares University, Mazandaran, Iran

2 School of Biology, College of Science,Tehran University,Tehran, Iran

Correspondence: A Abedian Kenari, Department of Fisheries, Faculty of Natural Resources and Marine Sciences,Tarbiat Modares versity, PO Box 46414-356, Noor, Mazandaran, Iran E-mail: aabedian@modares.ac.ir

Uni-Abstract

The e¡ects of supplemental dietary

phosphatidylcho-line (PtCho) on the growth performance, survival

and digestive enzyme activity of Caspian brown trout

(Salmo trutta caspius Kessler1877) alevins were

inves-tigated in this study Alevin (initial weight

0.8 0.12 g) was fed for 5 weeks with an

isoproteic-and isolipidic-formulated diet with increased levels of

PtCho from 0 to 60 g kg 1dry matter and

decreas-ing levels of soybean oil The increase in dietary

PtCho up to 4% led to an increase in alevin ¢nal

weight, suggesting that moderate PtCho levels are

needed during this stage of Caspian brown trout

Survival was not a¡ected by the dietary PtCho level

Phosphatidylcholine incorporation into the diet

caused higher phospholipase A2-speci¢c activity

Phosphatidylcholine did not show a bene¢cial e¡ect

on the speci¢c activity of amylase and protease

Li-pase-speci¢c activity was signi¢cantly higher in the

PtCho groups compared with the control group The

hepatosomatic index (HSI) was signi¢cantly

in£u-enced by the dietary PtCho level The results of the

present study indicated that the dietary

supplemen-tation of PtCho in the diet of Caspian brown trout

ale-vin improved growth and lipolytic enzyme activity

Keywords: Caspian brown trout, larval feeding,

phospholipid, digestive enzyme, growth

Introduction

The bene¢cial e¡ects of dietary phospholipids (PL) on

optimal growth, prevention of skeletal deformities,

stress resistance and survival of larval and juvenilestages of many species of marine ¢sh had been de-monstrated (Tocher, Bendiksen, Campbell & Bell2008) This bene¢cial e¡ect of PL may then be ex-plained by the numerous roles, including increaseddigestion and absorption of neutral lipid (Koven,Kolkovski, Tandler, Kissil & Sklan 1993; Salhi, Her-nandez-Cruz, Bessonart, Izquierdo & Fernandez-Pa-lacios 1999), synthesis and secretion of lipoproteins(Fontagne¤, Geurden, Esca¡re & Bergot 1998) and ei-cosanoids (Tocher et al 2008) Evidence suggests that

de novo PL biosynthesis pathways occur in ¢sh(Tocher 1995) However, it is unlikely that ¢sh cansynthesize PL at a rate su⁄cient to meet the forma-tion of metabolic requirements during the rapidgrowth of early development (Kanazawa, Teshima &Sakamoto 1985) and may depend on exogenous PL tofully satisfy the demand for lipoprotein synthesis(Fontagne¤, Burtaire, Corraze & Bergot 2000).Phosphatidylcholine (PtCho) is the major PL in ¢sh(Tocher et al 2008) This superiority in nutritional ef-

¢cacy may be explained by the speci¢c role of PtCho

as the major constituent of polar lipids in membranes(Coutteau, Kontara & Sorgeloos 2000), as well as itsspeci¢c function for the synthesis and secretion of li-poproteins (Salhi et al 1999), which play an impor-tant role in the transport of lipid As occurs in mostvertebrates, PtCho is generally the predominantphosphoglyceride class (95% of the total PL in lipo-protein) in ¢sh lipoproteins (Lie, Sandvin & Waagb1993; Field & Mathur 1995) Inclusion of soybeanPtCho in diets of common carp (Cyprinus carpio) in-creased total lipid digestibility (Fontagne¤ et al 1998);this could be related to an enhancement in the phos-

pholipase A2 (PLA2) activity in view of the

preferen-tial a⁄nity of the enzyme for PtCho (Iijima,Tanaka &

Ota 1998) Moreover, dietary PtCho could be a

poten-tial source of choline, which is regarded as a vitamin

for ¢sh (Halver 2002) Choline de¢ciency resulted in

the depletion of other methyl donors (Zeisel 1993), as

well as fatty in¢ltration of the liver and membrane

disruption in some ¢sh and shell¢sh species (NRC

1983) Kanazawa (1993) reported that PtCho seems

to be more e¡ective in promoting growth than

phos-phatidylinositol in larval Japanese ounder

(Para-lichthys olivaceus)

The Caspian brown trout, Salmo trutta caspius, is

an anadromous ¢sh and one of the nine sub-species

of brown trout in the world (Quillet, Faure,

Chevas-sus, Kreig, Harache, Arzel, Metailler & Boeuf 1992),

which is endangered and presently being considered

for a conservation programme in southern part of

Caspian Sea (Kiabi, Abdoli & Naderi 1999) Currently,

cultivated stocks are being reared for enhancement,

and protection of wild populations and the

mainte-nance of this species depend on the stocked ¢sh

origi-nating from aquaculture systems More recently, due

to ¢llet quality, the Caspian brown trout has attracted

interest for cage culture and raceways in Iran (Sarvi,

Niksirat, Mojazi Amiri, Mirtorabi, Ra¢ee & Bakhtiyari

2006)

Despite the importance of Caspian salmon as an

endangered species, little is known about its

nutri-tional needs, especially during the early growth

stages

The aim of the present study was to examine the

e¡ect of dietary PtCho on the incorporation of

di-gested lipid, growth and the activity of the digestive

enzymes: lipase, PLA2, amylase and protease of

Cas-pian brown trout alevins

Material and methods

Experimental ¢sh and diets

Alevin stage Caspian brown trout were brought from

the breeding and cultivation centre of Kelardasht,

Iran Four groups of ¢sh (mean initial weight

0.8 0.12 g) each were distributed randomly into

tri-plicate ¢breglass tanks (80 42  16 cm; 50 L) at a

density of 2 alevin per litre supplied with 15 0.5 1C

recirculated freshwater at a rate of 4.5 L min 1, with

a bio-¢lter The tanks were siphoned once a day

be-fore the ¢rst feeding and approximately 20% of the

water in each tank was replaced daily After 2 weeks’

holding under these conditions and feeding with a

commercial feed, each group was fed the tal diet manually for a period of 5 weeks.Water qual-ity parameters were maintained as follows:temperature, 15 0.5 1C; dissolved oxygen, 6.2

experimen-mg L 1; salinity,  1g L 1; ammonia-N and trite-N were 0.09 and 0.16 mg L 1; and pH, 7.6 Dur-ing the experiment, the diurnal cycle was 12 h light/

ni-12 h dark Fish were fed the experimental diets sixtimes daily: 08:00, 10:00, 12:00, 14:00, 16:00 and18:00 hours Survival was calculated by the indivi-dual counting of all the surviving larvae at the begin-ning and the end of the experiments

Four practical semi-puri¢ed ¢sh meal-based dietswere identical in their crude protein (57%) and lipid(17%) but di¡ered in their lipid composition andadded lipid component Puri¢ed soybean PtCho(90%, AppliChem, Darmstadt, Germany) was added

at four levels (0, 20, 40 and 60 g kg^1) by reducing bean oil The formulations and proximate composi-tions of the experimental diets are shown in Table 1.The dietary dry ingredients were weighed andground (100mm) and then mixed thoroughly Fishoil, soybean oil and soybean PtCho were added tothe appropriate diet and mixed again To obtain ap-propriate particle sizes, diets were sieved with 500and 700mm meshes, packed and stored at  20 1Cuntil feeding

soy-Proximate analyses of dietsThe total lipids of liver and diet samples were ex-tracted by homogenization in chloroform/methanol(2:1, v/v) according to Folch, Lees and Stanley (1957).Crude protein was determined using the Kjeldahlmethod (N 6.25) using an automatic Kjeldahl sys-tem (230-kjeltec Analyzer, Foss Tecator, H˛gans,Sweden) Dry matter in the diets was measured grav-imetrically after oven drying of homogenized sam-ples for 24 h at 105 1C and ash (incineration at 550 1Cfor 6 h) (Association of O⁄cial Analytical Chemists1995) The calculation of the gross energy of the dietswas carried out according to the NRC (1993) proce-dure, based on 1g crude protein 5 23.6 kJ, 1g crudefat 5 39.5 kJ and 1g carbohydrate 517.2 kJ Estimates

of the digestible energy (kJ) content in feed were culated using the default apparent coe⁄cients of di-gestibility of 0.9, 0.85 and 0.8 for crude protein, crudefat and carbohydrates (NFE) respectively (Moreau,Arredondo, Perraud-Gaime & Roussos 2003) Eachmeasurement was repeated ¢ve times If the coe⁄-cient of variation within these repetitions exceeded0.4%, the measurements were repeated

cal-In£uence of dietary phospholipid on Caspian brown trout A A Kenari et al Aquaculture Research, 2011, 42, 655^663

Fatty acid analysis of diets

Fatty acid methyl esters samples were analysed using

a Philips PU 4400 gas chromatograph (Cambridge,

UK) equipped with a fused silica capillary column

BPX-70 (25 m 0.32 mm, ¢lm thickness 0.25 mm)

and a FID detector The carrier gas was helium The

temperature programme included a gradient from

160 up to 230 1C, with a rate of increase of

1.5 1C min 1 Fatty acid methyl esters were identi¢ed

by known puri¢ed standards and quanti¢ed using a

response factor to internal and external fatty acidstandards The ¢nal values were an average of thethree replicate injections (Table 2)

The survival (S), hepatosomatic index (HSI), lipide⁄ciency ratio (LER), feed conversation ratio (FCR)and speci¢c growth rate (SGR) of the alevins werecalculated using the following formulas:

S ¼Number of fish in each group remaining on day 35

Initial number of fish  100

HSI¼100 Hepatopancreas wet weight

Body wet weight

SGR¼100 ðln final weight  ln initial weightÞ

Experimental durationðdayÞ

FCR¼ Dry feed intakeðgÞWet weight gainðgÞ

LER¼Total live weight gain

Total lipid fed

Table 1 Composition and proximate analyses of the

experi-mental diets containing di¡erent phosphatidylcholine levels

Lantern¢shes meal, Iran.

wTuna ¢sh oil, Havorash, Boushehr, Iran.

zAppliChem, Germany With the purity of 90%.

of diet): Fe, 4500 mg; Cu, 500 mg;

Co, 50 mg; Se, 50 mg; Zn, 6000 mg; Mn, 5000 mg; I, 150 mg;

cho-line chloride, 150 000 mg; career up to 1kg.

kAntioxidant: butylated hydroxytoluene.

Binder: Amet binder (component: crude protein: 71.98%,

crude ¢bre: 0.9%, ash: 17.8%, moisture: 9.55%).

(crude protein1crude lipid1¢bre1ash1moisture).

Gross energy was calculated using physiological fuel values of

re-spectively (Garling & Wilson 1976).

Table 2 Fatty acid composition (% of total fatty acids) of the experimental diets PtCho 0, PtCho 2, PtCho 4 and PtCho 6

Fatty acid

Experimental diets (PtCho %)

PtCho 0 PtCho 2 PtCho 4 PtCho 6

mono-Aquaculture Research, 2011, 42, 655^663 In£uence of dietary phospholipid on Caspian brown trout A A Kenari et al.

Digestive enzyme assays

At the end of the feeding trial (after a 2-day

starva-tion), the alevins were collected for growth analyses

and an enzyme assay Samples of enzymatic analysis

were washed in cold distilled water and stored in

li-quid nitrogen Five alevins per replicate were

dis-sected as described by Cahu and Zambonino Infante

(1994) on a glass maintained on ice (0 1C) Samples of

the entire digestive tract were homogenized

immedi-ately in 35 mg mL 1cold 10 mM PMSF and 0.1 M

phosphate bu¡ers (pH 7.5) with a homogenizer

(WIG-GENHAUSER, Berlin, Germany), followed by

centri-fugation (13,500 g; 30 min at 4 1C) The homogenate

was kept frozen ( 80 1C) until the enzymatic

deter-minations All the assay techniques were based on

photometric procedures in which the rate of

disap-pearance of the substrate or the rate of formation of

the product was measured Lipase-speci¢c activity

was assayed according to Mongklthanaruk and

Dharmosthiti (2002) using p-nitrophenyl myristate

as a substrate The reaction mixture contained

90mL of solution A (0.062 g of p-nitrophenyl

myris-tate in 10 mL of 2-propanol), 810mL of solution B

(0.4% triton X-100 and 0.1% gum Arabic in 50 mM

Tris-HCl, pH 8.0) and 100mL of a homogenized

en-zyme sample The product was detected at 410 nm

wavelength after incubation for 15 min at 37 1C

Pro-tease activity was measured with casein as the

sub-strate according to the method of Anson (1938)

slightly modi¢ed by Mongklthanaruk and

Dhar-mosthiti (2002) Brie£y, 1mL of 1.5% casein solution,

pH 7.0, was placed at 37 1C and, then,1mL of a diluted

enzyme sample was added The reaction was

incu-bated for 10 min before the addition of 2 mL of 0.4 M

trichloroacetic acid The solution with precipitates

was ¢ltered, and to 0.5 mL of the clear ¢ltrate, 2.5 mL

of 0.4 M Na2CO3 and 0.5 mL of Folin reagent were

added After further 10 min of incubation, the colour

density developed was determined at 660 nm

Amy-lase activity was assayed using starch as a substrate

(Bernfeld 1955) Brie£y, 100mL of properly diluted

en-zyme was added to a tube containing 0.5 mL of 1%

(w/v) starch solution The reaction mixture was

incu-bated at 37 1C for 15 min Then, 0.5 mL of

3,5-dinitro-salicylic acid was added to the mixture and boiled for

10 min The colour density was determined

spectro-photometrically at 540 nm Phospholipase A2 was

measured with PtCho as the substrate according to

the method of Price III (2007) Brie£y, enzyme

sam-ples were diluted in cold saline containing 2 mM

HEPES at pH 7.5 Twenty microlitres of each sample

mixture, 180mL of an assay mixture containing

5 mM triton X-100, 5 mM PtCho, 2 mM HEPES,

10 mM calcium chloride and 0.124% (w/v) mol blue dye in water were added Methanol was 5%

bromothy-in the ¢nal assay mixture The plate was immediatelyanalysed at 620 nm The incubation temperatures oflipase, amylase and protease were considered to beoptimal for enzymatic assays in mammals but al-lowed to compare the relative enzymatic adaptation

to the dietary treatment of each alevin group Solubleprotein was determined using the Bradford (1976)procedure with bovine albumin as a standard Theactivity of all enzymes was expressed as speci¢c ac-tivity being micromole of substrate hydrolysed perminute per milligram protein (U mg 1protein), andwas measured using a UV/VIS spectrophotometer(Shimadzu UV/VIS Spectrophotometer, Kyoto, Japan)

Statistical analysisData were analysed using one-way analysis ofvariance (ANOVA) Normality and homogeneity ofvariances were tested initially using Kolmogorov^Smirnov and Levene tests, respectively, and signi¢-cant di¡erences were determined using Duncan’smultiple comparison test All statistical analyseswere performed using the softwareSPSSversion 13.5(SPSS, Chicago, IL, USA) for Windows Po0.05 wasregarded as statistically signi¢cant

ResultsThe formulation and proximate composition of alldiets are shown in Table 1 The total lipid and proteincontents of all the diets were relatively constant Table

2 shows the fatty acid composition of the total lipids

in the diets Phosphatidylcholine supplementationsigni¢cantly increased the relative proportions of thedietary fatty acids 18:2n-6, 16:0 and 16:1 while de-creasing 14:0, 18:0 and 18:1n-9, 18:3n-3, 20:4n-6,20:5n-3 and 22:5n-3 in the diet (Table 2) Also,PtCho-incorporated diets were characterized by ahigher level of polyunsaturated fatty acid (PUFA)mainly linoleic acid (18:2n^6), although the highlyunsaturated fatty acid content was higher in PtCho 0

At the end of the experiment, the survival of alldietary treatments was high (98^100%) and not sig-ni¢cantly (P40.05) a¡ected by the dietary PtCho le-vels The ¢nal weights of Caspian brown trout alevinvaried between 2.87 and 3.19 g and were signi¢cantly(Po0.05) higher in treatments fed the PtCho 4 andIn£uence of dietary phospholipid on Caspian brown trout A A Kenari et al Aquaculture Research, 2011, 42, 655^663

PtCho 6 diets than the PtCho 0 and PtCho 2 diets

(Po0.05) (Table 3)

The addition of soybean PtCho to the diet resulted

in a signi¢cantly (Po0.05) higher SGR and a lower

FCR (Table 3) The lipid contents of the whole body

and liver were signi¢cantly (Po0.05) increased with

increasing PtCho supplementation Also, the HSI was

in£uenced by PtCho inclusion, as an increase in the

dietary PtCho level resulted in a signi¢cantly higher

HSI (0.9^1.39%)

Lipid e⁄ciency was not signi¢cantly a¡ected

(P40.05) by the addition of PtCho (Table 3) However,

¢sh fed the diets containing PtCho generally

achieved greater lipid e⁄ciency compared with the

control group

The activity of all enzymes increased compared

with the previous start feeding trial The speci¢c

ac-tivities of amylase and protease were not

signi¢-cantly di¡erent among the treatments (P40.05),

although the increase in dietary PtCho led to a slight

decrease in protease-speci¢c activity The alevin

group fed PtCho 0 had the highest amylase-speci¢c

activity The higher levels of lipase-speci¢c activity

were observed in alevin fed the PtCho-incorporated

diets Signi¢cant di¡erences (Po0.05) were observed

in PLA2-speci¢c activity; the highest speci¢c activity

was 32.9 m U mg 1protein for the PtCho 4 group As

shown in Fig 1, PLA2 and lipase activity reached a

plateau at 4% and 2% PtCho respectively

Discussion

The increase in dietary PtCho up to 4% led to a

sig-ni¢cant increase in alevins’ ¢nal weight and

im-proved the FCR Phospholipid is the major

component of cell biomembranes Therefore, thesupplemented PtCho diets may have increasedgrowth by supplying preformed PtCho to the ¢sh,thereby reducing the energy normally utilized inthe biosynthesis of PtCho (Craig & Gatlin III 1997)

In sea bream, Sparus aurata larvae, Hadas (1998)showed that PtCho have a stimulatory e¡ect on seabream larval feeding, while this was not observedwith phosphatidylethanolamine Koven, Parra,Kolkovski and Tandler (1998) reported that larvaefed a PtCho-supplemented micro diet had 35% high-

er ingestion rates compared with the ted micro diet in 21^26-day-old gilthead larvae.Other studies suggested that dietary PtCho has also

unsupplemen-a postprunsupplemen-andiunsupplemen-al physiologicunsupplemen-al in£uence unsupplemen-as well, curring in parallel or in tandem with its appetite-sti-mulating properties (Webster & Lim 2002) Thegrowth-enhancing e¡ect of PtCho in addition to itsrole in cell membrane structure and increasing in-gestion rate may have been due to the supply of cho-line The choline could have been obtained through

oc-a breoc-akdown of PtCho by phospholipoc-ase D, oc-as well

as from the base exchanges of PL (Gong, Lawrence

& Jiang 2003) Choline de¢ciency resulted in poorgrowth and fatty liver in rainbow trout (Rumsey1991) and cobia, Rachycentron canadum (Mai, Xiao,Ai,Wang, Xu, Zhang, Liufu & Ren 2009) Gong et al.(2003) reported that dietary PtCho could e¡ectivelyprovide the choline requirement in Litopenaeus van-namei, whereas Geurden, Radˇnz-Neto and Bergot(1995) showed that growth promotion and preven-tion of malformation e¡ects of PtCho were not mi-micked by choline Further investigations arerequired to better understand the role of the PtCho

in choline provision In the present study, thegrowth rates were probably not signi¢cantly in-

Table 3 Growth, survival and body lipid content of Caspian brown trout alevin fed diets containing varying levels of PtCho

Growth parameters

Experimental diets (PtCho %)

PtCho, phosphotidylcholine; SGR, speci¢c growth rate; FCR, feed conversion ratio; LER, lipid e⁄ciency ratio; HSI, hepatosomatic index Aquaculture Research, 2011, 42, 655^663 In£uence of dietary phospholipid on Caspian brown trout A A Kenari et al.

creased in ¢sh fed the PtCho 6 due to the reduced

level ratio of n-3:n-6 series fatty acids in these diets

(from 0.8 to 0.4; Table 2) The n-3 PUFA are required

at higher concentrations in ¢sh diets compared with

n-6 PUFA (Sargent, Bell, McEvoy, Tocher & Estevez

1999) The present study showed that alevin

survi-val was not a¡ected by PtCho, which is in agreement

with a similar previous experiment (Geurden,

Ber-got, Schwarz & Sorgeloos 1998; Hamza, Mhetli,

Khe-mis, Cahu & Kestemont 2008)

Our results showed that higher dietary PtCho led

to signi¢cantly higher liver lipid, HSI and whole body

lipid, while there was a non-signi¢cant (P40.05)

in-crease in LER Dietary PLs inin-creased whole-body

(Poston 1991; Coutteau et al 2000; Liu, Caballero,

Izquierdo, El-Sayed Ali, HernaŁndez-Cruz, Valencia &

FernaŁndez-Palacios 2002) and liver lipid

concentra-tions (Craig & Gatlin III 1997; Liu et al 2002) in

sev-eral species The reduced hepatopancreas volume in

PtCho 0 and 2 groups could have resulted from the

accumulation of lipid in the intestine through

insu⁄-cient PLs, a¡ecting the normal transportation

pro-cess of lipids from the enterocyte zone to other bodyparts In studies with common carp larvae, Fontagne¤

et al (1998) showed that dietary soybean PtCho vented intestinal steatosis and resulted in a larger li-ver volume and a larger hepatocyte volume Salhi

pre-et al (1999) found that dipre-etary PL contributes to protein production, thereby enhancing the e⁄ciency

lipo-of lipid transport from the digestive tract to the bodytissues

At the end of the experiment, the speci¢c activity

of all surveyed enzymes was signi¢cantly higher,which may be related to anatomical and physiologi-cal modi¢cations in the alevin Several researchescon¢rmed that the development stages signi¢cantlyin£uence the digestive enzyme activity in di¡erent

¢sh species (Buddington & Doroshev 1986; Kuz’mina1996) The speci¢c activity of lipase was signi¢cantlyhigher in PtCho-incorporated diets, albeit no cleardi¡erences in patterns were observed between diet-ary treatments The increase in lipase activity inthese groups may be explained by di¡erences in thefatty acid composition of the diet Iijima et al (1998)

diet (% PtCho) b

a

0 20 40 60 80 100 120

a

0 200 400 600 800 1000 1200 1400 1600 1800

Diet (% PtCho) b

0 20 40 60 80 100 120 140 160 180

Figure 1 E¡ect of dietary phosphatidylcholine on the speci¢c activity of amylase, lipase, phospholipase A2 and protease

in Caspian brown trout alevin Di¡erent values of enzyme activity (mean SD, n 5 5) with di¡erent superscript lettersare statistically signi¢cant (Po0.05)

In£uence of dietary phospholipid on Caspian brown trout A A Kenari et al Aquaculture Research, 2011, 42, 655^663

found that the speci¢city of pancreatic lipase activity

is related to both the acyl chain length and the degree

of unsaturation, which has been shown to have a

higher preference for PUFA as substrates As shown

in Table 2, PtCho incorporation into alevin diet

causes a signi¢cantly higher level of PUFA fatty acids

A speci¢c lipase activation has already been reported

in sea bass larvae (Morais, Cahu, Zambonino-Infante,

Robin, Rnnestad, Dinis & Conceicao 2004) and

Senegalese sole larvae (Morais, Caballero, Conceicao,

Izquierdo & Dinis 2006), when the diet was

supple-mented with di¡erent lipid sources Moreover, this

enzymatic adaptation response has been reported in

mammals (Brannon 1990)

Phospholipase A2 secreted into the intestinal tract

catalyses the hydrolysis of the fatty acid ester bond at

the sn-2 position of dietary and biliary PLs, resulting

in the formation of lysoglycerophospholipids and free

fatty acids (Iijima, Nakamura, Uematsu & Kayama

1990) The increase in PtCho caused a signi¢cant

in-crease in PLA2-speci¢c activity, particularly in the

PtCho 4 and PtCho 6 groups Phospholipase A2 and

lipase activity was found to increase in red drum

(Sciaenops ocellatus) larvae (Buchet, Zambonino

In-fante & Cahu 2000) and European sea bass larvae

(Zambonino Infante & Cahu 1999) fed diets

contain-ing increascontain-ing rates of respective dietary substrates,

i.e PLs and triglycerides We observed that lipolytic

enzyme-speci¢c activity increased with the dietary

supplementation PtCho The activity of lipases

strongly depends on the type and the concentration

of the surfactant (Moza¡ar, Weete & Dute 1994) and

also on how the lipophilic substrate is presented to

the enzyme (Brockman 1984) Phosphatidylcholine

may increase pancreatic enzyme secretion by

in-creasing lysophospholipids, which act as

supplemen-tary emulsi¢ers in the intestine of the alevin The

occurrence of a plateau in the lipolytic enzyme

activ-ity might indicate that there is a maximal capacactiv-ity of

lipolytic enzyme synthesis, as reported previously by

Zambonino Infante and Cahu (1999)

Amylase- and protease-speci¢c activity was the

same among the treatments, which may be due to

the constant substrate content in the diet Wold,

Hoehne-Reitan, Cahu, Zambonino Infante, Rainuzzo

and Kjrsvik (2007) reported the same observation

In contrast with other previous studies (Zambonino

Infante & Cahu 1994; Buchet et al 2000), which

re-ported that amylase-speci¢c activity is high during

the early larval stages and decreases during larval

development, amylase activity increased markedly

(twofold)

ConclusionsThe present study showed that the growth perfor-mance of Caspian brown trout alevin could be en-hanced by the addition of dietary soybean PtCho.Our experiment also showed that PtCho did signi¢-cantly a¡ect lipase- and PLA2-speci¢c activity Thesee¡ects of PtCho might be explained by increasing li-pid emulsi¢cation and the physiological role of PtCho

in lipoprotein synthesis and lipid transportation.With regard to growth performance, survival anddigestive enzyme activity, the optimal supplementa-tion of PtCho for Caspian brown trout was 4% (drymatter)

AcknowledgmentsThe authors wish to thank the Tarbiat Modares Uni-versity for their ¢nancial support Thanks are alsodue to Prof Dr Bill Koven for his kind help

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transduc-Aquaculture Research, 2011, 42, 655^663 In£uence of dietary phospholipid on Caspian brown trout A A Kenari et al.

Two strategies to unravel gene expression responses

larvae

Torunn Forberg1, Augustine Arukwe2& Olav Vadstein1

1 Department of Biotechnology, Norwegian University of Science and Technology,Trondheim, Norway

2 Department of Biology, Norwegian University of Science and Technology,Trondheim, Norway

Correspondence: T Forberg, Department of Biotechnology, Norwegian University of Science and Technology, Sem Sealandsveg 6/8, N7491 Trondheim, Norway E-mail: torunn.forberg@biotech.ntnu.no

Abstract

The commensal bacteria in the intestine play essential

roles in the development and functionality of the host

To unravel the host^microbe interactions in Atlantic

cod (Gadus morhua L.) larvae, we used two molecular

approaches: (1) suppression subtractive

hybridization-polymerase chain reaction (SSH-PCR) to identify host

gene responses and (2) expression analysis of selected

genes reported to be di¡erentially expressed in

gnoto-biotic zebra¢sh in a previous study to determine

whether these host responses are also conserved

in cod Suppression subtractive hybridization-PCR

identi¢ed 156 transcripts putatively regulated by the

presence of bacteria However, out of 22 selected

tran-scripts, only four were signi¢cantly di¡erentially

ex-pressed when quanti¢ed using quantitative (real-time)

PCR Expression analysis of selected genes from

zebra-¢sh revealed possible conservation of host responses

for three out of eight genes analysed For most of the

genes quanti¢ed, the gene expression pattern varied

between two biological replicates This may re£ect

dif-ferences in the bacterial composition in the rearing

bottles, and denaturing gradient gel electrophoresis

analysis con¢rmed signi¢cant di¡erences between the

two replicates with regard to bacterial diversity The

varying e¡ects on gene expression caused by

di¡er-ences in the microbial composition show the necessity

of further studies where axenic cod larvae are

com-pared with larvae raised in de¢ned and controlled

(gnotobiotic) environments

Keywords: bacteria-free, host response, gene

ex-pression, cod larvae, suppression subtractive

hybri-dization PCR

IntroductionSuccessful aquaculture of Atlantic cod (Gadus mor-hua L.) is still hampered by low survival at the larvalstage Opportunistic bacteria are thought to be amajor cause of these problems (Vadstein, ie, Olsen,Skjermo, Salvesen & Skjk-Brkg 1993) During in-tensive culture, the immature cod larvae are exposed

to, and interact with, large numbers of bacteria Theyactively drink water before yolk sac re-absorption,and the uptake of bacteria exceeds the drinkingrate by two orders of magnitude (Reitan, Natvik &Vadstein 1998) As a consequence, the undi¡eren-tiated intestinal tract is exposed to a large number ofbacteria, even before the start of exogenous feeding.The host^microbe interactions in the gut of the codlarvae can lead to the formation of a healthy stableintestinal micro£ora or to infection and disease(Hansen & Olafsen 1999; Olafsen 2001) Whether abacterium will colonize the intestine is determined

by interactions between the di¡erent bacteriapresent, nutrient availability, adhesion propertiesand cross talk with the host cells (Kelly, Conway &Aminov 2005; Corthesy, Gaskins & Mercenier 2007)

In intensive rearing of marine ¢sh larvae, the search focus is now shifting from non-speci¢c removal

re-of bacteria in the rearing water to controlling andmaintaining a bene¢cial micro£ora (Ring & Birkbeck1999; Skjermo & Vadstein 1999; Vine, Leukes & Kaiser2006) However, there is still a lack of knowledgeconcerning the host^microbe interactions that takeplace during the ¢rst weeks of larval growth, and thesubsequent formation of an intestinal micro£ora.The use of gnotobiotic vertebrates (containing

a known, de¢ned microbial £ora) has revealed that

microbial colonization directly a¡ects a wide range of

biological processes, including nutrient processing

and adsorption, development of the mucosal

im-mune system and epithelial proliferation (Rawls,

Sa-muel & Gordon 2004; Smith, Mccoy & Macpherson

2006; Cheesman & Guillemin 2007) A gnotobiotic

model used to investigate the gene responses to the

micro£ora in zebra ¢sh (Danio rerio) revealed 212

host genes whose expressions were regulated by

bac-teria (Rawls et al 2004) However, zebra¢sh hatch at a

fairly developed state and are phylogenetically

dis-tant from marine ¢sh

The aim of this study was to investigate the e¡ect of

bacterial presence on the di¡erential gene expression

patterns of cod larvae We have established a protocol

for bacteria-free rearing of cod larvae, making it

possi-ble to compare cod larvae grown without bacteria

with those grown in a mixed bacterial community (T

Forberg, O Vadstein & A Arukwe, unpublished data)

To investigate host gene expression responses, we

chose two strategies: (1) suppression subtractive

hybri-dization- polymerase chain reaction (SSH-PCR) to

gen-erate sequences of di¡erentially expressed genes, as an

unbiased approach to identify host responses, and (2) a

biased approach, expression analysis of selected genes

reported to be di¡erentially expressed in gnotobiotic

zebra¢sh (Rawls et al 2004), to determine whether

these host responses are also conserved in cod

Materials and methods

Biological material and experiments

Cod eggs were disinfected twice with 400 ppm

glutar-aldehyde for 10 min (Salvesen & Vadstein 1995;

Salvesen, ie & Vadstein 1997), and hatched in ¢ltered

(0.22mm Micropores

, Derbyshire, UK), autoclaved water (FASW), containing 10 ppm each of rifampicin

sea-and ampicillin (T Forberg, O Vadstein & A Arukwe,

unpublished data) The water temperature during

disinfection was around 6 1C; during the experiment,

this temperature was increased by 11day 1up until

12 1C All work was performed using sterile

equip-ment under a laminar £ow hood After hatching, the

cod larvae were transferred to (Nalgenes, Thermo

Scienti¢c, Rochester, NY, USA) rearing bottles,

con-taining either 2 L free (FASW) or

bacteria-containing seawater The bacteria-bacteria-containing

sea-water used was aged seasea-water, generated by ¢ltering

seawater through a GF/F (Whatmans, GE

Health-care, Amersham, UK) ¢lter to remove large particles,

and stored for 2 weeks without aeration at room

tem-perature (approximately 20 1C) before use Aged water that had been UV treated for 5 min was alsoused, to achieve variation with regard to the bacteriapresent K-selected bacteria will presumably dominatethe aged seawater, while UV treatment will lead to adomination of r-strategists (Andrews & Harris 1986;Skjermo, Salvesen, ie, Olsen & Vadstein 1997).Bacteria-free rotifers to be used as feed were obtainedaccording to the protocol of Tinh, Phuoc, Dierckens,Sorgeloos and Bossier (2006), with one modi¢cation:the rotifer eggs were left to hatch in 10 ppm of rifampi-cin and ampicillin Bacteria-free rotifers were added tothe cod rearing bottles from day 3 until day 17 posthatch Axenic Isochrysis sp was also added, in accor-dance with the green-water technique (Skjermo & Vad-stein 1993) Dead larvae were removed and counted ondays 4,10,12,14 and17.The cod larvae were reared untilday 17 post hatch On day 17, they were sacri¢ced usingMS-222 (0.5 g L 1, lethal dose), rinsed in MilliQ waterand placed in RNAlaterssolution (Ambions, LifeTech-nologies, Carlsbad, CA, USA) for storage at  20 1C.Two separate start feeding experiments were per-formed: the ¢rst to generate cod samples for SSH-PCRand the second to generate samples for gene expres-sion analysis of cod genes identi¢ed by SSH and codhomologues of genes selected from the zebra¢sh study

sea-In the ¢rst experiment, three bacteria-free rearingbottles and four bacteria-exposed (two with aged sea-water and two with UV-treated aged seawater) werestocked with 80 larvae L 1 In the second experiment,two bacteria-free (BF1 and BF2) and two bacteria-exposed (M1 and M2) (containing aged seawater)rearing bottles were stocked with 30 larvae L 1 (alower density of larvae was chosen to reduce theamounts of bacteria-free rotifers needed)

Evaluation of bacteria-free conditions,bacterial density and diversity

Samples from the cod rearing water and from rotiferand algae cultures were taken every other day of theexperiments Liquid and solid M65 media (consisting

of 0.5 g peptone, 0.5 g tryptone and 0.5 g yeast extract,dissolved in 800 mL FASW and 200 mL MilliQ water)and Marine Broth (DifcoTM, BD, Franklin Lakes, NJ,USA) were used to check for bacterial contamination.Serial dilution plating was used to estimate the den-sity of culturable bacteria in the rearing bottles con-taining aged seawater

In the second start feeding experiment, £ow metry was used to investigate the presence and den-sity of bacteria in all cod rearing bottles Brie£y, SYBRAquaculture Research, 2011, 42, 664^676 Gene-expression responses to bacteria in cod larvae T Forberg et al.

cyto-green (SYBR Green I, Molecular Probes) was added

to water from the rearing bottles, and a FACSScan

£ow cytometer (Becton Dickinson, BD) was used

to detect £uorescent particles (Marie, Brussaard,

Thyrhaug, Bratbak & Vaulot 1999) Filtered

auto-claved seawater was used to quantify the number of

background particles Flow cytometry counts were

performed on days 6, 10 and 17 after hatching

Denaturing gradient gel electrophoresis (DGGE)

was used to investigate the diversity of the microbial

community present in the two bacteria-containing

cod rearing bottles (M1 and M2) in the second start

feeding experiment DNA was isolated from

centri-fuged 10 mL water samples taken on days 10 and 17

post hatch, using the Qiagen DNAeasy kit (Hilden,

Germany) according to the manufacturer’s protocol

PCR was performed using 16S rDNA primers

338f-GC and 517r (Muyzer, De Waal & Uitterlinden 1993),

under the following conditions: initial denaturation

at 95 1C for 4 min, followed by 40 cycles of 30 s at

95 1C, 60 s at 50 1C and 90 s at 72 1C and a ¢nal

elon-gation step for 30 min at 72 1C A denaturing gradient

of 35^60% was used, and the gel was run for 17 h at a

voltage of100 V (using the Ingeny phorU system) The

DGGE gel was stained with SYBR Green SYBR Gold

(InvitrogenTM, Life Technologies, Carlsbad, CA, USA)

for 30 min and photographed under UV light

Dena-turing gradient gel electrophoresis gel images were

analysed using theGEL2Ksoftware (provided by Svein

Norland, Department of Biology, University of

Ber-gen, Norway) Peak detection parameters were set to

2 for vertical and horizontal sensitivity and ¢ve-pixel

smoothing was used The relative bandwidth was set

to 0.0003 The peak area matrix for the samples was

exported and normalized to per cent of sum area

Pearson’s correlation coe⁄cients were calculated to

compare the normalized band intensity pro¢les

be-tween samples The Shannon index (Shannon 1948)

and the relative diversity J0(evenness) were used as

measures of diversity in the DGGE pro¢les

Generation of subtracted library and

sequence analysis

Suppression subtractive hybridization-PCR was

per-formed under contract by EcoArray (Alachua, FL,

USA), using polyA cDNA from pooled larvae samples

(n 511) from the bacteria-free replicates and the

bac-teria-exposed replicates (n 5 8) from the ¢rst start

feeding experiment The experiment was performed

in both forward and reverse directions to obtain two

clone libraries containing up- and down-regulated

genes respectively Sequenced clones were analysedusing Blastx against the GenBank protein databaseand Blastn against the GenBank nucleotide database.The e-value cut-o¡ was set at 10 5for blast searches.EST sequences were submitted to the NCBI GenBankEST database and assigned accession numbersGW574323^GW574464, while ribosomal and mito-chondrial sequences were submitted to the GenBanknucleotide database (acc# GU931777^GU931790)

RNA isolation and cDNA synthesisCod larvae from the second start feeding experimentwere placed in TRK lysis bu¡er (supplied with theE.Z.N.Askit) and B-mercaptoethanol before homo-genization with a rotor-stator Total RNAwas isolatedusing the E.Z.N.Astotal RNA kit (Omega Bio-Tek,Norcross, GA, USA) according to the manufacturer’sprotocol, including on-membrane DNase I treatment.Larvae were pooled to reduce the e¡ect of inter-indi-vidual variation on gene expression RNA was iso-lated from two pools of ¢ve larvae for each of thebacteria-containing replicates (M1 and M2), and twopools of ¢ve, plus one with four larvae from the bac-teria-free (BF1) rearing bottle RNA concentrationwas measured using a NanoDrops

ND-1000 UV ble Spectrophotometer (NanoDrop Technologies,Wil-mington, DE, USA), and RNA integrity was con¢rmed

visi-by inspection of intact ribosomal 28S and 18S bandsafter denaturing gel electrophoresis

Total cDNA for qPCR was generated from 1mg totalRNA for all samples, using a mixture of random andpoly-T primers from the iScript cDNA synthesis kit(Bio-Rad, Hercules, CA, USA) according to the manu-facturer’s protocol A control lacking reverse tran-scriptase enzyme was included in each run Thesynthesized cDNA was diluted 1:6 before qPCR

Primer design, ampli¢cation e⁄ciency andquantitative PCR

Twenty-two sequences identi¢ed from the subtractedlibraries (generated from the ¢rst start feeding ex-periment) were selected for qPCR Speci¢c primerswere designed to verify the di¡erential expression ofthese genes in cod larvae from experiment 2.Based on the ¢ndings of Rawls et al (2004) andhighly similar sequences available from cod in Gen-Bank, qPCR primers were designed to speci¢callyamplify eight genes (Table 1) Serum ameloid A1 wasone gene reported as regulated by bacteria in zebra-

¢sh, but as there was no similar sequence availableGene-expression responses to bacteria in cod larvae T Forberg et al Aquaculture Research, 2011, 42, 664–676