Aquaculture research, tập 41, số 5, 2010

E-mail: lconcei@ualg.pt Abstract Despite the recent progress in the production of inert diets for ¢sh larvae, feeding of most species of interest for aquaculture still relies on live fee

This issue is the result of the Special Session entitled

‘‘Basic And Applied Aspects Of Aquaculture

Nutri-tion: Healthy Fish For Healthy Consumers’’ carried

out on 17^18 September 2008 in Krakow, Poland as

part of the Annual Meeting of the European

Aqua-culture Society This session was co-sponsored by

the Organization for Economic Cooperation and

Development (OECD) Co-operative Research

Pro-gramme on Biological Resource Management for

Sustainable Agriculture Systems, Trade and

Agricul-ture Directorate In particular we appreciate help in

the organization of the session by Dr Carl Christian

Schmidt The European Aquaculture Society

co-sponsored the session and help from the Executive

Director, Dr Alistair Lane, need to be recognized

The Ohio State University, School of Environment

and Natural Resources, contributed ¢nancially to

the session

One of the primary goals of ¢sh nutrition is to

pro-duce healthy food for human consumption This can

only be achieved via a thorough understanding of

¢sh nutritional requirements and the appropriate

choice of feed ingredients that will secure the

eco-nomic e⁄ciency and sustainability of the industry,

i.e decreased use of ¢shmeal and replacement

with plant protein and lipid sources The conference

intended to promote and focus the research on

aquaculture nutrition by making a link to the human

food chain

The conference addressed the major areas of basic

aspects of ¢sh nutrition by gathering experts in

aqua-culture nutrition that could outline the current state

of knowledge in the ¢eld, and present coherent

per-spectives on the improvements needed in ¢sh culture

to ful¢ll public expectations in terms of healthy food

and its sustainable production Sustainable ¢sh

pro-duction must concentrate on the use of plant protein

and lipid sources in ¢sh diets to replace ¢shmeal

re-sulting in healthy seafood from the human point of

view Consequently, pollution originating from

¢sh and other aquatic animal farming must be

decreased This will result in higher economic

e⁄ciency of seafood production as the cost of ¢sh

food (diets) is the largest single cost (over 50%) ofproduction

From the very beginning of the session concept, sults of the conference were to be transferred to pro-fessionals and therefore the subject of the regularpeer-review process The intent was also to dissemi-nate the results to the general public and the media

re-in the form of a brochure that will summarize the jor conclusions of the conference (abstracts) with thereference materials to each of the contributors, its

ma-¢eld of expertise and current professional activity.The session was also thought to contribute to publicdebate by focusing on the production of healthy hu-man food at a time while the world ¢shery is shrink-ing and thus move from exploitation to sustainableproduction As outlined in the recent FAO ‘‘State ofthe World Aquaculture 2006’’document, the sessionintended to cross national and institutional bound-aries and establish a framework for the large-scaledevelopment of aquaculture

That was truly a forum for multidisciplinary actions The programme areas that were covered byinvited speakers can be grouped into the followingmajor topics: (1) digestive tract morphology and regu-lation of nutrient uptake, (2) endocrine and neuralregulation of food intake, (3) molecular biology tools

inter-to validate and compliment biological nutrition data,(4) major nutrient requirements, (5) replacement of

¢shmeal protein and lipids with plant ingredients,(6) speci¢c nutritional needs for di¡erent life stages

of ¢sh, from larvae to broodstock nutrition and (7)management of aquaculture waste

There are a number of challenges that must beovercome to maintain acceptable growth rates andfeed e⁄ciency values at higher levels of substitution

of ¢shmeal The ¢rst is cost of plant protein trates The second challenge facing the aquafeed in-dustry as it moves to substitute higher amounts of

concen-¢shmeal with plant proteins pertains to known tritional limitations of plant proteins The third chal-lenge to overcome to developing plant protein-basedaquafeeds for intensively grown ¢sh speciesconcerns unknown nutrients and biologically active

nu-materials in ¢shmeal that are not present in plant

protein concentrates and that may be necessary

diet-ary constituent for optimum growth and health of

¢sh These three applied aspects of ¢sh nutrition were

reviewed but undoubtedly deserve special session in

the near future as the logical consequence of the

ba-sic nutrition research These applied aspects will be

tackled with powerful new molecular biology tools,not only in a descriptive manner, but also by path-way-speci¢c metabolomic markers in ¢sh nutritionstudies

Konrad DabrowskiRonald Hardy

r 2010 The Authors

REVIEW ARTICLE

Live feeds for early stages of fish rearing

Lu|¤s E C Conceicao1, Manuel Yu¤fera2, Pavlos Makridis3, So¢a Morais1& Maria Teresa Dinis1

1

Center for Marine Sciences ^ CCMAR, University of Algarve, Faro, Portugal

2 Instituto de Ciencias Marinas Andaluc|¤a, CSIC, Apartado O¢cial, Puerto Real, Spain

3 Institute of Aquaculture, Hellenic Center for Marine Research, Heraklion, Crete, Greece

Correspondence: L E C Conceicao, CCMAR, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal E-mail: lconcei@ualg.pt

Abstract

Despite the recent progress in the production of inert

diets for ¢sh larvae, feeding of most species of interest

for aquaculture still relies on live feeds during the

early life stages Independently of their nutritional

value, live feeds are easily detected and captured,

due to their swimming movements in the water

column, and highly digestible, given their lower

nu-trient concentration (water content480%) The

pre-sent paper reviews the main types of live feeds used

in aquaculture, their advantages and pitfalls, with a

special emphasis on their nutritional value and the

extent to which this can be manipulated The most

commonly used live feeds in aquaculture are rotifers

(Brachionus sp.) and brine shrimp (Artemia sp.), due to

the existence of standardized cost-e¡ective protocols

for their mass production However, both rotifers and

Artemia have nutritional de¢ciencies for marine

spe-cies, particularly in essential n-3 highly unsaturated

fatty acids (HUFA, e.g., docosahexaenoic acid and

ei-cosapentaenoic acid) Enrichment of these live feeds

with HUFA-rich lipid emulsions may lead to an excess

dietary lipid and sub-optimal dietary protein content

for ¢sh larvae In addition, rotifers and Artemia are

likely to have sub-optimal dietary levels of some

ami-no acids, vitamins and minerals, at least for some

species Several species of microalgae are also used

in larviculture These are used as feed for other live

feeds, but mostly in the‘green water’ technique in ¢sh

larval rearing, with putative bene¢cial e¡ects on

feeding behaviour, digestive function, nutritional

va-lue, water quality and micro£ora Copepods and

other natural zooplankton organisms have also been

used as live feeds, normally with considerably better

results in terms of larval survival rates, growth andquality, when compared with rotifers and Artemia.Nonetheless, technical di⁄culties in mass-producingthese organisms are still a constraint to their routineuse Improvements in inert microdiets will likely lead

to a progressive substitution of live feeds However,complete substitution is probably years away formost species, at least for the ¢rst days of feeding

Keywords: microalgae, rotifers, Artemia, pods, nutritional value, ¢sh larvae

cope-IntroductionLive feeds are the main item in the diet of cultured

¢sh larvae and they are of particular importancewhen rearing marine ¢sh larvae of the altricial type.Altricial larvae are those that remain in a relativelyundeveloped state until the yolk sac is exhausted At

¢rst-feeding the digestive system is still rudimentary,lacking a stomach, and much of the protein digestiontakes place in hindgut epithelial cells (Govoni, Boeh-lert & Watanabe 1986) Such a digestive system is inmost cases incapable of processing formulated diets

in a manner that allows survival and growth of thelarvae comparable to those fed live feeds In fact, de-spite recent progress in the development of inert dietsfor ¢sh larvae (e.g., Lazo, Dinis, Holt, Faulk & Arnold2000; Cahu & Infante 2001; Koven, Kolkovski, Hadas,Gamsiz & Tandler 2001), feeding of most species of in-terest for aquaculture still relies on live feeds duringthe early life stages Even the ‘Artemia replacement’products increasingly used in commercial operationsare normally used in co-feeding with live feeds (e.g.,

Curnow, King, Partridge & Kolkovski 2006;

Vega-Orellana, Fracalossi & Sugai 2006; Hamza, Mhetli &

Kestemont 2007; Rosenlund & Halldo¤rsson 2007)

However, the low digestive capacity of altricial

lar-vae might not be the only aspect responsible for them

normally requiring live feed Live preys are able to

swim in the water column and are thus constantly

available to the larvae Most formulated diets tend to

aggregate on the water surface or, more commonly,

sink within a few minutes to the bottom, and are thus

normally less available to the larvae than live feeds

In addition, since larvae are believed to be ‘visual

fee-ders’, adapted to attack moving prey in nature, the

movement of live feed in the water is likely to

stimu-late larval feeding responses Finally, live prey, with a

thin exoskeleton and a high water content (normally

4 80%), have a lower nutrient concentration and may

be more palatable to the larvae once taken into the

mouth, compared with the hard, dry formulated diet

This last point is rather critical as any feed item must

enter the mouth whole, i.e., feed particles have to be

smaller than the larva’s mouth gape, and are quickly

accepted or rejected on the basis of palatability

(Fer-naŁndez-D|¤az, Pascual & Yu¤fera 1994; Bengtson 2003)

The present paper aims to review the main types of

live feeds used in aquaculture, their advantages and

pitfalls, with a special emphasis on their nutritional

value and the extent to which this may be

manipu-lated It also reviews the main concerns and potential

bene¢ts regarding the micro£ora composition of live

feed when it is added to larval tanks Finally, it

dis-cusses the constraints of using live feeds to study ¢sh

larvae nutritional requirements, the possibilities of

using tracer studies to overcome such constraints

and assessment of the future of live feeds in ¢sh larvae

production

Microalgae

Main utilizations of microalgae

Microalgae constitute the ¢rst link in the oceanic

food chain, i.e., the primary producer, due to its

abil-ity to synthesize organic molecules using solar

en-ergy In aquaculture, microalgae are produced as a

direct food source for various ¢lter-feeding larval

stages of organisms such as bivalve molluscs (clams,

oysters and scallops), the larval stages of some

mar-ine gastropods (abalone) and early stages of penaeid

shrimp larvae (Yu¤fera & LubiaŁn 1990) They are also

used as an indirect food source, in the production of

zooplankton (e.g., rotifers and Artemia), which in

turn is used as food for the carnivorous larvae ofmany of the marine ¢sh and shrimp species presentlyfarmed Finally, intensive rearing of bivalves has sofar relied on the production of live microalgae, whichcomprises on average 30% of the operating costs in abivalve hatchery

For rearing marine ¢sh larvae according to the

‘green water technique’, algae are used directly inthe larval tanks This technique is nowadays a nor-mal procedure in marine larviculture, given that ithas been widely reported to improve ¢sh larvalgrowth, survival and feed ingestion (e.g., ie, Makri-dis, Reitan & Olsen 1997; Reitan, Rainuzzo, ie &Olsen 1997) The observed larval quality enhance-ment when using microalgae in the rearing waterhas been explained by di¡erent studies, whichshowed that microalgae seemed to provide nutrientsdirectly to the larvae (Mo¡att 1981), to contribute

to the preservation of live prey nutritional quality(Makridis & Olsen 1999), to promote changes in thevisual contrast of the medium and in its chemicalcomposition (Naas, Naess & Harboe 1992; Naas, Huse

& Iglesias 1996) and to play an important role in themicro£ora diversi¢cation of both the tank and thelarval gut (Nicolas, Robic & Ansquer 1989; Reitan

et al 1997; Skjermo & Vadstein 1999; Olsen, Olsen,Attramadal, Christie, Birkbeck, Skjermo & Vadstein2000) More recently, Rocha, Ribeiro, Costa and Dinis(2008) showed that ¢sh larvae feeding ability is alsoin£uenced by the presence of microalgae in the tank.However, this e¡ect is not the same among speciesand has been shown to be more pronounced withgilthead seabream than with Senegalese sole larvae(Rocha et al 2008)

Main species of microalgaeThe ¢rst microalgae species produced for aquacul-ture were selected from those produced naturally inpioneering ¢sh farms and were probably the easiestones to cultivate (Muller-Fuega, Moal & Kaasa2004) However, other species were later investigatedbased on their biological characteristics and perfor-mance under laboratory culture conditions, as well

as on their nutritional and energetic properties.Among the most important selection criteria formicroalgae, the following can be highlighted:

1 cell size appropriate to the demands of the mer organisms;

consu-2 adequate nutritional value;

3 high digestibility;

Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

4 ease of culture at high densities;

5 short life cycle, reproducible in captivity; and

6 tolerance to environmental variations

Using these selection criteria, 16 genera of

microal-gae are generally produced nowadays Still, some

species dominate and it is possible to relate such

spe-cies to their corresponding utilization Class

Bacci-lariophyceae (Diatoms) are usually given to bivalve

molluscs and crustacean larvae as they are rich in

silicates, which constitute their cell walls (frustules)

and are necessary for bivalves and crustaceans for

the formation of rigid structures Classes

Prasinophy-ceae (e.g., Tetraselmis suecica, Tetraselmis Chuii) and

Chrophyceae (e.g., Dunaliella tertioleta, Chrorella

min-utissima) are ideal food for crustacean larvae, when

complemented by Baccilariophyceae for silicate

sup-ply Class Prymnesiophyceae (e.g., Isochrysis galbana)

is widely used to culture marine ¢sh larvae (Brown

1991), while class Thraustochytriidae (e.g.,

Schizoch-trium sp.), which consists of heterotrophic chromists,

has mostly been used as feed for live prey species

(Brachionus sp and Artemia sp.)

Microalgae product types

A general feature of marine microalgae is their high

polyunsaturated fatty acid (PUFA) content However,

the availability of microalgae as an a¡ordable PUFA

source is limited At present, many ¢sh farms have

their own facilities to produce microalgae for use

during the ¢rst feeding of marine ¢sh and crustacean

larvae The investment in such facilities is high and

can represent 30% of a hatchery operating cost

(Coutteau & Sorgeloos 1992) In addition,

productiv-ity may be variable depending on the season

New products and methodologies with better

cost-e¡ectiveness have been investigated and developed

This includes microcapsules, dried microalgae,

yeasts or yeast-based diets, bacteria,

thraustochy-trids (Knauer & Southgate 1999; Langdon & nal

1999) and algal pastes (Heasman, Diemar, O’Connor,

Sushames & Foulkes 2000) A fast-growing range of

such commercial products is available, including live

microalgae concentrates, and frozen and freeze-dried

microalgae Results using these products are

gener-ally good For instance, centrifuged concentrates of

Pavlova lutheri in combination with Chaetoceros

calci-trans or Skeletonema costatum yielded 85^90% of the

growth of a mixed diet of live microalgae for oyster

larvae Saccostrea glomerata (Heasman et al 2000)

Hatcheries that already have the infrastructure for

algal mass production may also prepare their ownconcentrates on-site, and thus limit their algal pro-duction to less busy periods of the season, bettermanage their requirements for microalgae and alsoreduce costs due to over-production (Knuckey,Brown, Robert & Frampton 2006)

Techniques for growing microalgaeVarious techniques have been developed to grow mi-croalgae on a large scale, ranging from less con-trolled extensive to monospeci¢c intensive cultures.However, the controlled production of microalgae isstill a complex and expensive procedure Culture ofmicroalgae for aquaculture purposes (rearing of mol-lusc, shrimp and ¢sh larvae) takes place mostly on-site, i.e., in the ¢sh farms where they are utilized,although a new industry is emerging for the produc-tion of microalgae and delivery in lyophilized, frozen

or other form to the farms (Navarro & Sarasquete1998; Muller-Fuega et al 2004) There are variousmethods for the culture of microalgae, but the induc-tion of ‘blooms’ by fertilization (addition of nutrients

to the culture medium) is the most usual In ¢shfarms, stock cultures are kept under controlled condi-tions and protected from contamination by other mi-croalgae, ciliates and potentially harmful bacteria.Up-scaling of the microalgae cultures takes place inseveral steps, and the ¢nal large-scale cultures are of-ten located outdoors under natural light conditions.Cultures may be monospeci¢c or polyspeci¢c Poly-speci¢c cultures are normally carried out in open airtanks, or ponds, with water volumes exceeding

100 m3, while monospeci¢c cultures are carried out

Photo-bioreactors are a relatively recent advance

for the production of microalgae in aquaculture

farms They enable the production of large biomasses

of microalgae in high-cell-density cultures, in a small

area and with a low input of labour Tubular

(serpen-tine and manifold) and £at-plate bioreactors are

commonly used for this purpose Small volumes of

high-density microalgae are easier to handle by ¢sh

farmers but present some problems as well, as they

require the use of experienced personnel and all

microalgae production is dependent on a few

cul-tures Such devices may need a cooling system, as

heat is accumulated in the system, and automated

addition of carbon dioxide

In relation to the timing of harvest of microalgae

during the culture period, culture strategies can be

divided into: (i) batch cultures, where no growth

medium is added after initial inoculation; and

semi-continuous cultures, where a part of the culture is

harvested and new growth medium is added

subse-quently, several times during the culture period (Ii,

Hirata, Matsuo, Nishikawa & Tase 1997) According

to Fogg (1975), the limiting factors for microalgae

duction by photoautotrophy, resulting from the

pro-duction method, are the exhaustion of nutrients,

reduction in illumination due to the increase in cell

density (shadowing) and the inhibition of cell

divi-sion due to the accumulation of catabolites However,

the various factors may be interdependent and a

parameter that is optimal for one set of conditions is

not necessarily optimal for another

Algal cultures need to be enriched with nutrients

in order to overcome the nutrient de¢ciencies of

sea-water This includes the macronutrients nitrate and

phosphate (in an approximate ratio of 6:1), silicate (if

growing diatoms) and micronutrients, comprising

various trace metals and the vitamins thiamin (B1),

cyanocobalamin (B12) and sometimes biotin The

Walne medium and the F/2 medium are the two most

extensively used enrichment media and are suitable

for the growth of most algae There are also

commer-cially available nutrient solutions that are suitable for

mass production of microalgae in large-scale

exten-sive systems These solutions contain only the most

important nutrients and are made of

agriculture-grade rather than laboratory-agriculture-grade fertilizers

Heterotrophic culture may provide a cost-e¡ective,

large-scale alternative method of cultivation for some

microalgae that utilize organic carbon substances as

their sole carbon and energy source This mode of

growth eliminates the requirement for light and,

therefore, o¡ers the possibility of considerably

in-creasing the microalgal cell concentration and,hence, volumetric productivity in batch systems Inthe last decade, knowledge of the cultivation of het-erotrophic marine algae that accumulate PUFAs hasincreased However, knowledge and production ofsuch products are limited and restricted only to veryfew companies (Muller-Fuega et al 2004) Further-more, as heterotrophic algae are not used directly asfeed in the aquaculture industry, their performance

is not known and is yet to be determined Two trophic species that are commonly used in live foodenrichment emulsions or in ¢sh diets, due to theirhigh levels of docosahexaenoic acid (22:6n-3; DHA),are the dino£agellate Crypthecodinium cohnii and thefungal thraustochytrid, Schizochytrium sp (De Swaarf,Pronk & Sijtsma 2003)

hetero-Nutritional value of microalgaeWhenever microalgae are used as a direct foodsource or as an indirect food source, in the produc-tion of rotifers, Artemia or copepods, growth of theanimals is usually superior when a mixture of sev-eral microalgal species is used (Becker 2004) Thisprobably occurs as di¡erent species compensate oneanother for eventual de¢ciencies in given nutrients.Special care is needed when selecting microalgae forongrowing live feeds for marine ¢sh larvae, in order

to avoid the nutritional de¢ciencies of the latter, inparticular in terms of n-3 highly unsaturated fattyacids (HUFA)

The dry matter composition of microalgae is highlyvariable, even within a given species, with proteincontents ranging from 12% to 35%, lipid from 7.2%

to 23% and carbohydrates from 4.6% to 23% (Becker2004) The protein content of microalgae is a majorfactor in determining its nutritional value and maychange considerably with the composition of thegrowing medium (Becker 2004) Nitrogen concentra-tion seems to be particularly important in this respect.De¢ciencies in the n-3 HUFA contents of microal-gae may cause severe mortalities and/or quality pro-blems in shrimp, mollusc and marine ¢sh larvae Inaddition, such de¢ciencies may also cause reducedfecundity of rotifer and copepod cultures Signi¢-cant concentrations of eicosapentaenoic acid (EPA;20:5n-3) are normally present in diatom species(C calcitrans, Chaetoceros gracilis, S costatum andTha-lassiosira pseudonana), Nannochloropsis sp., T suecica,Tetraselmis chuii, D tertioleta and C minutissima(Brown 1991; Becker 2004) High concentrations ofLive feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

DHA are found in I galbana and P lutheri (Brown

1991; Becker 2004) and particularly in

Thraustochy-triidae (e.g., Schizochtrium sp.), which can contain

over 70% of its weight as lipids and have a DHA

con-tent up to 35% of their total fatty acids (Sijtsma & de

Swaarf 2004)

Microbial aspects in phytoplankton cultures

During the culture of microalgae, a high organic load

is progressively accumulated, which becomes the

substrate for the proliferation of bacteria Bacterial

cells may attach to microalgae cells or may proliferate

in the water Bacteria associated with microalgae

may reach very high values of culturable bacteria,

such as 108mL 1or 103cell 1 (Salvesen, Reitan,

Skjermo & ie 2000) In batch cultures, the level of

bacteria is lower during the exponential phase and

reaches a maximum during the stationary phase

(Salvesen et al 2000; Makridis, Alves Costa & Dinis

2006) In semi-continuous cultures, where there is a

periodic replacement of part of the culture by new

growth medium, numbers of bacteria tend to

stabi-lize at a value lower than that observed in similar

batch cultures Unsuccessful culture of microalgae,

characterized by low growth rates and an extended

lag phase of the cultures, may result in a high

bacter-ial load (Nicolas et al 1989; Salvesen et al 2000) The

cultured microalgae cells and the bacterial

commu-nities associated with the microalgae cultures are in

constant interaction, resulting either in the

suppres-sion of growth of speci¢c groups of bacteria or in

de-creased growth of the cultured microalgae (Munro,

McLean, Barbour & Birkbeck1995; Fukami, Nishijima

& Ishida 1997; Suminto & Hirayama 1997; Kokou,

Ferreira, Tsigenopoulos, Makridis, Kotoulas,

Magou-las & Divanach 2007) The outcome of these

interac-tions may depend on the method of microalgae

production, the microalgae species grown, the

growth media used, the quality of seawater and the

growth phase of the culture (Salvesen et al 2000;

Makridis et al 2006)

Antimicrobial activity has been detected in

ex-tracts of microalgae (Du¡ & Bruce 1966; Austin &

Day 1990; Austin, Baudet & Stobie 1992; Tendencia &

dela Pena 2003) and in bacteria isolated from

micro-algae (Makridis et al 2006) This antimicrobial

activ-ity can be caused by

(i) associated microbiota (Makridis et al 2006);

(ii) antimicrobial proteins or fatty acids produced by

the microalgae cells (Kokou et al 2007); or

(iii) free oxygen radicals produced due to the synthetic activity of the microalgae cells(Marshall, de Salas, Oda & Hallegraef 2005).Examination of the bacterial populations present inmicroalgae cultures using molecular approachesable to detect non-culturable and culturable bacter-ial strains revealed a di¡erent picture than studies

photo-of the culturable microbiota Cultures photo-of P lutheri,

I galbana, C calcitrans, S costatum, C gracilis andChaetoceros muelleri harboured a broad spectrum

of species belonging to the groups of teria,b-Proteobacteria, g-Proteobacteria, Cytophaga^Flavobacterium^Bacteroides (CFB) bacteria group,Actinobacteria and Bacillus Members of the Roseo-bacter clade and the CFB group were dominant inthe microalgae cultures In microalgae cultures, cul-turable members of the Vibrio group were absent orpresent in very low numbers (Salvesen et al 2000;Tendencia & dela Pena 2003; Sainz-Hernandez &Maeda-Martinez 2005; Makridis et al 2006)

a-Proteobac-Addition of microalgae to the rearing tanks of ine ¢sh larvae has a positive e¡ect on the growth andsurvival of the larvae It has been suggested that thispositive e¡ect may be due to the bacteria associatedwith the microalgae cultures (Reitan, Rainuzzo, ie

mar-& Olsen 1993) However, evidence for this is still weakand further studies are needed

RotifersMain utilization of rotifersSince the 1970s, the rotifers and more speci¢callyBrachionus plicatilis constitute an essential part ofthe feeding during the larval stages of marine ¢shand crustaceans (Yu¤fera 2001; Lubzens & Zmora2003) Its body size (between 70 and 350mm depend-ing on the strain and age) makes this organism an ap-propriate prey to start feeding after the resorption ofvitelline reserves of many species In fact, Brachionus

is widely used as ¢rst food during a period of days orweeks depending on the reared species, being re-placed afterwards by a larger prey species, usuallyArtemia nauplii (Yu¤fera, Rodr|¤guez & LubiaŁn 1984;Polo, Yu¤fera & Pascual 1992; Olsen et al 2000) Ob-viously, ¢sh species having a wider mouth gape at theonset of feeding may start directly on Artemia nauplii.Besides the above-mentioned body size feature, themain advantages of this organism to be used as liveprey in hatchery large-scale production are the fol-lowing: (i) a high population growth rate, (ii) feeding

by ¢ltration of particles in suspension, being able to

ingest microalgae, yeast, bacteria and organic

parti-cles, (iii) a good tolerance to culture conditions

and handling, something common in euryhaline

animals, and (iv) to exhibit an appropriate energy

content and reasonable nutritional value that, in

ad-dition, is relatively modi¢able by dietary

manipula-tion during routine feeding and/or by means of

speci¢c post-culture enrichment Another advantage

of this prey is that it can be made permanently

avail-able Rotifers are a renewable resource and the

pro-duction chain can be completely established in the

hatchery, avoiding the dependence on external

sup-plies The major inconveniences arise from the

occur-rence of episodic collapses as well as from the e¡ort

required for maintaining the whole plankton chain

Raising rotifers for larviculture requires the

pro-duction of large amounts of organisms with

appro-priate size and nutritional quality In order to meet

these objectives, many di¡erent strains have been

iso-lated from nature and acclimated to the laboratory

conditions In addition, di¡erent mass culture

techni-ques have been developed, and a variety of

microal-gae species, yeast and commercial products have

been tested and used as food during the routine

cul-ture and the following enrichment period

Main species and strains of rotifers

Variations in female’s body size were already observed

in the late 1970s, but were noted mainly during the

1980s when di¡erent morphotypes and strains were

described according to body size and spine shape The

di¡erent strains were grouped into large (L-type),

medium (SM-type) and small (S-type) Brachionus

pli-catilis (Yu¤fera 1982; Fukusho & Okaushi 1983; Snell &

Carrillo 1984; Fu, Hirayama & Natsukari 1991; Go¤mez

& Serra 1995) Currently, this group is considered to

be a multi-species complex of 9^15 di¡erent species

and biotypes This complex includes species formally

described as Brachionus plicatilis sensu stricto,

Brachionus rotundiformis, Brachionus ibericus and

Brachionus manjavacas, together with a series of

lineages discernible by molecular techniques (Segers

1997; Ciros-Pe¤rez, Go¤mez & Serra 2001; Go¤mez, Serra,

Carvalho & Lunt 2002; Suatoni,Vicario, Rice, Snell &

Caccone 2006; Fontaneto, Giordani & Serra 2007;

Mills, Lunt & Go¤mez 2007) Besides the formal

spe-cies, at least the lineages Brachionus sp Nevada,

Bra-chionus sp Cayman and BraBra-chionus sp Austria have

been identi¢ed as common in hatcheries (Papakostas,

Dooms, Triantafyllidis, Deloof, Kappas, Dierckens,

De Wolf, Bossier, Vadstein, Kui, Sorgeloos & poulos 2006; Dooms, Papakostas, Ho¡man, Delbare,Dierkens, Triantafyllidis, De Wolf, Vadstein, Abatzo-poulos, Sorgeloos & Bossier 2007; Kostopoulou &Vadstein 2007; Baer, Langdon, Mills, Schulz & Hamre2008) The clari¢cation of the taxonomical situationwill continue in the coming years From a practicalpoint of view, as a prey for larviculture, it is still useful

Abatzo-to use the identi¢cation of large (L), medium (SM) andsmall (S and SS) morphotypes referring to the relativebody size (Table 1)

Rearing techniques for rotifersSince the establishment of the basis for the culture ofBrachionus (Ito 1960), di¡erent techniques of massculture have been developed in order to obtain highand constant productions (see for instance: Hirata &Mori 1967; Theilacker & McMaster 1971; Hirata 1974;Yu¤fera & Pascual 1980; Gatesoupe & Robin 1981;Yoshimura, Hagiwara, Yoshimatsu & Kitajima 1996;Suantika, Dhert, Nurhudah & Sorgeloos 2000; Dhert,Rombaut, Suantika & Sorgeloos 2001; Park, Lee, Cho,Kim, Jung & Kim 2001; Lubzens & Zmora 2003; Olsen2004)

The Brachionus plicatilis complex of species andbiotypes reproduce mostly by parthenogenesis,although a sexual phase may occur under speci¢cenvironmental conditions The most relevant aspect

is its high fecundity, which allows a population cation time of 24^48 h (Hirayama & Kusano 1972;Hirayama, Watanabe & Kusano 1973; Yu¤fera, LubiaŁn

dupli-& Pascual 1983; Korstad, Olsen dupli-& Vadstein 1989) vided that the appropriate abiotic and feeding condi-tions are supplied, a single parthenogenetic female

Pro-Table 1 Accepted species (in bold) and other biotypes belonging to Brachionus plicatilis species complex grouped according to former size-related classi¢cation (Go¤mez et al., 2002; Baer et al., 2008)

L -biotypes SM -biotypes S and SS biotypes

B plicatilis B ibericus B rotundiformis

B manjavacas B ‘coyrecupiensis’ B ‘lost’

num-Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

may generate a large o¡spring in a few days The

rear-ing techniques developed for rotifers take advantage

of this characteristic of their reproductive biology

The Brachionus population exhibits an exponential

growth while the favourable conditions persist,

fol-lowed by a decrease and cessation in growth when

the food is exhausted or the chemical and

microbio-logical conditions fall out beyond tolerance ranges

Therefore, all the di¡erent culture systems that have

been developed attempt to maintain the exponential

growth by supplying high food levels and by

prevent-ing excessive accumulation of nitrogenous waste

The best descriptor of the health and growth status

of the cultured population is the egg ratio This index

is a direct indicator of fecundity, and therefore of the

potential for growth in the following hours (Snell &

Carrillo1984;Yu¤fera et al.1984) The best growth rates

occur approximately at a temperature between 20

and 35 1C, a salinity of 10^35 and a pH of 7.0^9.0

(Hirayama & Kusano 1972; Pascual & Yu¤fera 1983;

Miracle & Serra 1989; Yu¤fera & Navarro 1995) The

re-novation pattern of the culture media (water plus

al-gal cells) determines the basic methods, from batch

culture (no renovation) to semi-continuous culture

(partial renovation) and continuous culture

(perma-nent renovation) In addition, independent of the

cul-ture system, to establish a complete production chain,

three basic volume scales have to be considered: the

stock culture (50^500 mL) aiming to maintain the

genetic strain under optimal conditions; the starter

culture (from 5 to 50 L) proceeding directly from the

genetic strain and used to inoculate large volume

cul-tures; and the mass culture (from 50 L to several m3)

Obviously, this basic chain may change according to

the operating conditions of each hatchery

In the batch culture system, an inoculum with a

relatively low individual density is seeded in a dense

microalgae suspension and the rotifer population

grows for several days until the exhaustion of the

al-gal cells The growth pattern follows a sigmoid curve

and the maximum rotifer density attained increases

with an increase in the initial food concentration

fol-lowing a saturation response The whole production

is harvested at the end of the exponential phase and

used Commonly, a small part of this production is

used as a starter in the next production cycle two or

three times The quality of this starter (inoculum) is

decisive for the success of the rotifer culture The

per-iodic use of the starter coming directly from the stock

culture guarantees the health of the cultured

popu-lation and the stability of the production This is a

very common system in hatcheries, and many

exam-ples of its application may be found in the literature(Lubzens 1987; Dhert et al 2001)

In the semi-continuous system, harvesting andmedia renovation are frequent, and account for a no-table part of the total volume Harvested volumeranges from 10% to 50% and the harvesting fre-quency ranges from 1 to 3 days The culture may lastseveral weeks When the renovation frequency isevery 24 h or less and the renovated volume is con-stant, the culture can be associated with a continu-ous culture (Boraas 1983; Schluter, Soeder &Growneweg 1987) Like the batch system, the semi-continuous culture is widely used in larvicultureand is usually combined with batch culture (Hiraya-

ma & Nakamura1976; Suantika et al 2000; Lubzens &Zmora 2003; Olsen 2004)

The continuous rotifer culture systems are based

on the chemostat methodology (Droop & Scott 1978;James & Abu Rezeq 1989) In chemostat-like systems,the daily renovation rate is constant After the initialgrowth, the population reaches a steady state, main-taining an almost constant rotifer density duringweeks During the steady state, the growth rate isequal to the renovation rate The female density at-tained during the steady state, and consequently theproduction, depends on the food level (Boraas 1983;Schluter et al.1987;Walz1993; Navarro & Yu¤fera1998).The use of a concentrate microalgae paste, freeze-dried microalgae or commercial feeds allows higherlevels of cell and particle concentration to be attained

in the culture media than that normally obtainedwith microalgae suspension cultures (Hirayama &Nakamura 1976; Yu¤fera & Navarro 1995) This hasbeen the basis for the development of super-intensiveculture techniques in which the rotifer density andproduction are notably higher than that obtainedwith traditional methods (Yoshimura et al 1996; Fu,Ada, Yamashita, Yashida & Hino 1997; Suantika et al.2000; Park et al 2001; Suantika, Dhert, Rombaut,Vandenberghe, De Wolf & Sorgeloos 2001;Yoshimura,Tanaka & Yoshimatsu 2001) This kind of mass cul-ture is associated with continuous or similar sys-tems, with permanent addition of a high-quality dietthat ensures rapid rotifer growth Rotifers may invest

a large amount of energy in relation to their body mass in reproduction Therefore, failure in food sup-ply following a period of large investment in eggproduction may result in collapse of the rotiferculture Another important issue in relation to high-density production of rotifers is the removal of am-monia Several techniques have been suggested, such

bio-as lowering of pH, ion exchanger, membrane ¢lter

units or recirculation systems The daily harvest

de-pends considerably on the culture system and ranges

from 50 to 100 rotifers mL 1of culture in the batch

system up to 10,000 mL 1in the super-intensive

sys-tems Given that these culture systems are quite

vari-able but also somewhat £exible, each hatchery

usually develops a tailored system adapted to its

spe-ci¢c characteristics For instance, tanks may be quite

di¡erent in volume and shape, even in the same

facil-ity; the water pre-treatment varies depending on the

seawater source and quality; the Brachionus strain is

selected according to the ¢sh species being cultivated

and to the environmental conditions; and the kind of

food for mass rearing and for the ¢nal nutritional

enrichment is selected according to the production

necessities and the desired ¢nal biochemical

compo-sition, as well as the routine procedures for

harvest-ing and culture renovation

Feeding and enrichments for rotifers

Brachionus is a suspension feeder that ingests cells

and particles available in the water column by

¢ltra-tion Many di¡erent potential feeds have been tested

in order to have a high fecundity and/or to achieve an

appropriate biochemical composition.Yeast,

microal-gae and commercial feeds are regularly used either

for population growth or nutritional enrichment

Many microalgae species have been tested for rearing

Brachionus (Hirayama,Takaga & Kimura 1979; Yu¤fera

et al 1983; Lubzens 1987; Yu¤fera & LubiaŁn 1990) The

most commonly used are Nannochloris spp.,

Nanno-chloropsis spp.,Tetraselmis spp., P lutheri and I

galba-na Baker’s yeast Saccaromices cereviceae is also a

common and inexpensive food source for rotifer

pro-duction in hatcheries that started being used early on

(Hirata & Mori 1967; Yu¤fera & Pascual 1980; James,

Dias & Salman 1987; Nagata & White 1992), usually

in combination with microalgae Sprayed and

freeze-dried microalgae and concentrated microalgae

pastes are also commonly used (Hirayama &

Naka-mura 1976; Gatesoupe & Luquet 1981; Dhert et al

2001; Lubzens & Zmora 2003; Olsen 2004)

As in other zooplanktonic prey, rotifer biochemical

composition is of primary importance for larval

nu-trition The lipid and essential fatty acids pro¢le is

relatively modi¢able by dietary manipulation

(Ben-Amotz, Fishler & Schneller 1987; Lubzens, Tandler &

Minko¡ 1989; Rainuzzo, Reitan & Olsen1997) Baker’s

yeast and some microalgae species support high

po-pulation growth but the rotifers produced are

nutri-tionally de¢cient or poorly balanced in the lipidfraction as larval food In order to achieve an appro-priate content of essential fatty acids, rotifers can beenriched with microalgae (ie, Reitan & Olsen 1994;FernaŁndez-Reiriz & Labarta 1996; Hamre, Srivastava,Rnnestad, Mangor-Jensen & Stoss 2008), hand-made marine oil emulsions (Watanabe, Kitajima &Fujita 1983; Rodr|¤guez, Pe¤rez, Bad|¤a, Izquierdo, Her-naŁndez-Palacios & Lorenzo 1998; Bell, McEvoy, Este-vez, Shields & Sargent 2003; Hamre, Srivastava et al.2008), prepared microparticulated feeds (Walford &Lam 1987) or commercial products (FernaŁndez-Reiriz, Labarta & Ferreiro1993; Faulk & Holt 2005;Vil-lalta, Este¤vez & Bransden 2005; Hamre, Srivastava

et al 2008) A variety of enrichment protocols havebeen described, but overall a re-feeding or an enrich-ment period of 8^24 h results in the desired biochem-ical composition Nevertheless, feeding regimesbased on a combinetion of baker’s yeast and microal-gae or an adequate combination of two microalgalspecies (for instance: Tetraselmis sp or Nannochlorop-sis sp.1I galbana) during rotifer rearing can result in

an appropriate composition, with no need for furtherenrichment (Nagata & White 1992; Klaoudatos, Iako-vopoulos & Klaoudatos 2004) Furthermore, supply-ing microalgae directly to the larval rearing tankcontributes towards maintaining the nutritionalquality of rotifers up to their ingestion by the larvae.Rotifers can also be enriched with speci¢c com-pounds to perform experiments in ¢sh larvae nutri-tion, such as vitamins, iodine or selenium (Gime¤nez,Kotzamanis, Hontoria, Estevez & Gisbert 2007; Hamre,Mollan, Sle & Erstad 2008) The protein content andamino acid pro¢le is less modi¢able but may changedepending on the nutritional condition and repro-ductive status (Szyper 1989; Yu¤fera & Pascual 1989;Yu¤fera, Parra & Pascual1997; Makridis & Olsen 1999)

Nutritional valueDry matter content, caloric value and chemical com-position establish the nutritional value of rotifers,and are determined by size and nutritional state(Lubzens et al 1989; Go¤mez et al 2002; Lubzens &Zmora 2003; Baer et al 2008) According to Lubzensand Zmora (2003), rotifer’s protein content ranges be-tween 28% and 63%, lipid from 9% to 28%, and car-bohydrate from 10.5% to 27% of the dry weight (DW).The importance of rotifer HUFA contents in mar-ine ¢sh larvae nutrition has long been established,with numerous studies being available on rotifer lipidLive feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

enrichment procedures (see Lubzens et al.1989;

Rain-uzzo et al 1997; Lubzens & Zmora 2003, for detailed

reviews) Rotifer lipids have a high phospholipid

con-tent (34^43% of total lipid), and 20^55% of

triacyl-glycerols (Lubzens & Zmora 2003) However, when

cultured on yeast alone, rotifers are poor in HUFAs

required for normal development and good survival

of marine ¢sh larvae DHA, EPA and arachidonic acid

(20:4n-6; ARA) contents can, however, be

manipu-lated by short- or long-term feeding with selected

microalgae species, lipid emulsions, lipid-rich

micro-capsules and other feed types, as mentioned above

(Lubzens et al 1989; Rainuzzo et al 1997; Lubzens &

Zmora 2003)

Despite the high crude protein content of rotifers,

the availability of rotifer protein to marine ¢sh larvae

is of concern (Srivastava, Hamre & Stoss 2006)

Roti-fers are a common diet for ¢rst-feeding many marine

¢sh species, when the larval digestive system is still

largely immature (Govoni et al 1986; Rnnestad &

Conceicao 2005) It has been proposed that soluble

protein may be more easily digestible in young ¢sh

larvae (Carvalho, SaŁ, Oliva-Teles & Bergot 2004)

Sri-vastava et al (2006) established that soluble protein

makes up to 50.6% of the rotifer crude protein

Furthermore, growth is essentially protein

deposi-tion, and ¢sh larvae have very high amino acid

re-quirements to support fast growth and high energy

demands (Rnnestad, Tonheim, Fyhn, Rojas-Garcia,

Kamisaka, Koven, Finn, Terjesen, Barr & Conceicao

2003; Rnnestad & Conceicao 2005) In addition, the

amino acid pro¢le of rotifer protein has been shown

to be unbalanced for several ¢sh larval species

(Conceicao, Grasdalen & Ronnestad 2003; Aragao,

Conceicao, Dinis & Fyhn 2004; Saavedra, Conceicao,

Pousao-Ferreira & Dinis 2006; Saavedra, Beltran,

Pousao-Ferreira, Dinis, Blasco & Conceicao 2007)

Such unbalances may a¡ect larval performance and

quality

Rotifers seem to have high concentrations of

vita-mins C, E, B1 and B2 (van der Meeren, Olsen, Hamre

& Fyhn 2008) Rotifers cultured on commercial

en-richment products seem to meet the larval

require-ments for all the B-vitamins, with the possible

exception of thiamine (Hamre, Srivastava et al

2008) However, rotifers are possibly de¢cient in

sev-eral minsev-erals, including copper, iodine, zinc and, in

particular, manganese and selenium (Hamre,

Srivas-tava et al 2008) The last two were found in

consider-ably lower amounts compared with copepods (Table 2;

Hamre, Srivastava et al 2008), the natural diet of

marine ¢sh larvae

Microbial aspects in rotifer cultures

The microbiota of cultured rotifers is similar to themicrobiota of the water in the cultures (Skjermo &Vadstein 1993) Rotifers are ¢lter-feeders, and are able

to ¢lter bacteria from the surrounding medium stein, ie & Olsen 1993; Makridis, Fjellheim, Skjermo

(Vad-& Vadstein 2000) The bacteria associated with fers grown on fresh baker’s yeast plus oil varied from1.6 to 7.6 103 bacteria per rotifer, whereas thenumbers of culturable bacteria in the culture watermicrobiota ranged from 0.6 to 25 107CFU mL 1(Skjermo & Vadstein1993) The numbers of culturablebacteria in rotifer cultures grown on microalgaewere very low (about 100 CFU rotifer 1) in compari-son with rotifer cultures grown on yeast (ie et al.1994) The culturable microbiota in samples fromrotifers fed baker’s yeast with added capelin oilwas dominated by members of the Pseodomonas/Alca-ligenes group, Cytophaga/Flavobacterium group, Alter-omonas and Vibrio (Skjermo & Vadstein 1993).Culture-independent characterization of bacteriaassociated with rotifer cultures showed a di¡erentpicture where members of Rhodobacteraceae andArcobacter were dominant, whereasVibrio, Alteromo-nas and Roseobacter were also detected in rotifersgrown on commercial diets (McIntosh, Ji, Forward,Puvanendran, Boyce & Ritchie 2008) Analysis of mi-crobiota in rotifer cultures fed with Culture Selcosbydenaturing gradient gel electrophoresis showed thatbatch cultures showed continuous shifts in the domi-nant bands, whereas microbiota in rotifers produced

roti-in a recirculation system was far more stable baut, Suantika, Boon, Maertens, Dhert,Top, Sorgeloos

(Rom-& Verstraete 2001) The dominant bacterial speciespresent in this recirculation system belonged tothe genus Marinomonas and Pseudoalteromonas.The high bacterial load present in rotifers duringthe culture and enrichment process may cause pro-blems in the rearing of marine ¢sh larvae Oppor-tunistic bacteria present in the rotifers may bedetrimental for the larvae (Skjermo & Vadstein 1999).Rinsing of rotifers removes a large part of the bacteriathey carry However, bacteria brought into the rear-ing system may become a problem in later stages ofthe rearing Several approaches have been suggestedfor the removal of opportunistic bacteria, such as theuse of:

1 various chemicals and disinfectants wang & Muroga 1992);

(Tanasom-2 ultraviolet radiation (Munro, Henderson, Barbour

& Birbeck 1999);

3 ozone (Suantika et al 2001); and

4 live bacterial additives (probiotics)

Addition of probiotics in live food may have several

purposes: (i) inoculation of bacteria that have a

posi-tive e¡ect on the growth and survival of the live feed

organisms in culture (Ii et al 1997; Rombaut, Dhert,

Vandenerghe, Verschuere, Sorgeloos & Verstraete

1999; Douillet 2000; Dhert et al 2001); addition of

bacteria that limit the growth of opportunistic

bac-teria harmful for the Artemia and the larvae

(Gate-soupe, Arakawa & Watanabe 1989; Makridis 2000)

and (iii) addition of bacteria that may colonize the

¢sh larvae and may have a positive e¡ect on the

growth and survival rates of the larvae (Makridis,

Martins, Reis & Dinis 2008)

Artemia

Main utilizations ofArtemia

The brine shrimp Artemia, together with rotifers, are

the most widely used live preys in aquaculture As a

consequence of its high cost (due to low and

unreli-able natural resources and increased demand) and

low DHA content, as will be discussed below, a

popu-lar strategy in popu-larviculture of many marine species

has been to attempt early weaning in conjunction

with a prolonged rotifer feeding period to eliminate

the need to use Artemia Nonetheless, this is not

al-ways possible and in some species whose larvae are

relatively larger at hatching, Artemia might even be

the only live prey used in larviculture Its high larity in both aquaculture and aquarium pet tradestems mostly from its ease of handling and mass cul-ture One of the most interesting features of this or-ganism is its ability to form dormant cysts that arehighly resistant to adverse environmental condi-tions, can be kept viable for years (remarkable ‘shelf-life’) and are extremely convenient to transport, storeand use They are normally kept under dry (vacuum)and cool conditions and, when needed, they can besimply rehydrated in water, under favourable envir-onmental conditions, and hatch as a nauplii ino24 h The ease and simplicity of hatching brineshrimp nauplii makes them the most convenient andleast labour-intensive live foods available for aquacul-ture (Lavens & Sorgeloos 2000) The nauplii stage (in-star I) is still dependent on its endogenous reserves,its digestive tube is not yet completely formed andthe mouth and anus are closed After about 8 h, theanimal moults into the metanauplius I (instar II)stage, when it starts ¢lter-feeding small food particles(6.8^27.5mm, with an optimum of16.0 mm) (Van Stap-pen 1996a; Fernandez 2001) Metanauplius are con-tinuous non-selective ¢lter-feeding organisms, just

popu-as rotifers This is another important characteristic

of these organisms that was the basis for the ment of the bioencapsulation process (Van Stappen1996a), which enables enrichment with nutrients(mostly lipids, fatty acids and vitamins) or any othersubstances (hormones, chemotherapeutics or pro-phylactic products and vaccines, particularly if

develop-Table 2 Average biochemical composition of copepod nauplii, copepods, enriched rotifers and enriched Artemia, based on values published by (1) van der Meeren et al (2008), (2) Moren et al (2006), and (3) Hamre, Srivastava et al (2008)

DW, dry weight; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ARA, arachidonic acid; FAA, free amino acids.

Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

lipo-soluble), as long as the particles are of an

ade-quate dimension This characteristic is quite

impor-tant, given the biochemical composition of this prey,

which was selected more due to its convenience of

use than for its nutritional value for marine ¢sh

lar-vae The term bioencapsulation has, however, been

reviewed and should be avoided, as it later became

evident that Artemia are not mere passive carriers of

fatty acids, as will be discussed below

Main species ofArtemia

The brine shrimp Artemia is a relatively primitive

form of crustacean of the class Branchiopoda The

lack of a true carapace places them in the suborder

Anostraca, and further in the family Artemidae

There is still disagreement and controversy

regard-ing the taxonomy of the di¡erent Artemia species

Originally, taxonomists gave species names to

popu-lations with di¡erent morphologies, collected at

dif-ferent temperatures and salinities (Van Stappen

1996a), but this was later abandoned and most

aqua-culture-related literature only uses the genus

desig-nation Artemia spp Molecular techniques have been

recently used to clarify the phylogenetic

relation-ships of bisexual and parthenogenic Artemia species

and populations but this has not yet been completely

resolved (Baxevanis, Kappas & Abatzopoulos 2006;

Hou, Bi, Zou, He, Yang, Qu & Liu 2006) A multitude

of di¡erent species and strains of Artemia cysts can be

harvested along the shorelines of hypersaline lakes,

coastal lagoons and solar saltworks from all over the

world (Van Stappen 1996a), and eight species have

been documented in scienti¢c literature: three from

the NewWorld ^ Artemia franciscana, Artemia

persimi-lis and Artemia monica ^ and ¢ve in the Old World ^

Artemia salina, Artemia urmiana, Artemia sinica,

Artemia sp and Artemia tibetiana (Hou et al 2006)

These species and strains may di¡er considerably in

the diameter of cysts and the length of hatched

nau-plii (varying between 400 and just over 500mm),

hatching synchrony and e⁄ciency (normally 150^

250 000 nauplii are produced per gram of cysts),

growth rate, biochemical composition (essential fatty

acids, ascorbic acid, pigments and trace elements)

and thus nutritional value, but important di¡erences

can also be found within strains, depending on the

batch and harvesting, processing or storage

condi-tions (Van Stappen 1996a) Broadly speaking, and

from a nutritional point of view, Artemia spp may be

divided into a marine or a freshwater origin and these

two groups may be distinguished by their lipid andfatty acid composition, with marine-type strains pre-senting higher levels of total lipid and triacylglycer-ols, as well as higher levels of EPA and ARA andlower levels of linolenic acid (LNA; 18:3n-3) thanfreshwater-type strains (Navarro, Amat & Sargent1993)

Commercial harvesting of Artemia cysts has torically been performed in two major areas in theUnited States ^ Great Salt Lake (GSL), UT, and SanFrancisco Bay, CA However, from the mid-1980s on-wards, GSL cysts dominated the world market, and by

his-2000 over 90% of the cysts sold commercially wereharvested in this lake, creating a problem of overde-pendence of the world aquaculture industry on a sin-gle (and unreliable) source of this valuable resource.Other natural sources then started to be evaluatedfor their commercial potential, being located mainly

in Central Asia ^ Iran, China, Siberia and stan ^ and in Argentina Quite a few semi-natural ormanaged (resulting from deliberate inoculation ofArtemia in solar salt works) production sites are alsobeing exploited worldwide but their production is ty-pically quite low, only meeting the local demand (VanStappen 1996a; Lavens & Sorgeloos 2000)

Turkmeni-A common protocol in many marine hatcheriesworldwide is to feed the younger larvae with newlyhatched nauplii from selected strains and batchesthat produce small nauplii with a high EPA content,replacing them, as the ¢sh larvae are able to acceptlarger prey, by enriched Artemia metanauplii (GSL).After 12 and 48 h of enrichment, GSL Artemia meta-nauplii are typically around 660mm and 790 mm longrespectively (Merchie 1996)

Feeding and enrichments forArtemia

An important limitation of Artemia spp enrichment

as a tool to study larval nutritional quantitative andqualitative requirements is the notorious lack of con-sistency in this procedure, as considerable variabilityhas been reported in the essential fatty acid contentafter Artemia spp enrichment despite attempts tostandardize protocols (Merchie 1996) An importantfraction of the ¢ltered lipids is digested, assimilatedinto the Artemia body and metabolized, and not justsimply accumulated in the gut In addition to the dif-ferential metabolism of certain fatty acids, incorpo-rated fatty acids redistribute themselves among lipidclasses with high unpredictability, both during en-richment and particularly under starving conditions,

after being added to the larval rearing tanks

(Navar-ro, Henderson, McEvoy, Bell & Amat 1999) The high

variability in the ¢nal composition stems from the

fact that we are dealing with live organisms with an

active metabolism and physiology, particularly in the

early stages of development, which may also show

asynchronous development Their physiology can be

a¡ected by a multitude of exogenous (e.g.,

environ-mental, such as temperature, aeration,

hydrody-namics in the enrichment media, or size of micelles

in the emulsion) and endogenous (e.g., stage of

devel-opment and intrinsic metabolic rate) factors that

can-not be fully controlled or standardized (Navarro et al

1999)

A large variety of lipid enrichment products,

com-mercially available or ‘home made’, have been used

over the years Classically, lipid enrichment protocols

were developed using marine oil (mostly ¢sh oils)

emulsions, where tiny micelles (droplets) of

triacyl-glycerols are produced and can be ¢ltered from

the water This, however, tends to result in a prey

with high levels of neutral lipids and potentially

im-balanced protein/lipid ratios and an excess of

triacyl-glycerols (Sargent, McEvoy, Estevez, Bell, Bell,

Henderson & Tocher 1999; Morais, Conceicao,

Rn-nestad, Koven, Cahu, Infante & Dinis 2007) On the

other hand, a well-documented high larval

require-ment has been established for phospholipids and a

bene¢cial e¡ect of supplying essential fatty acids as

phospholipids, rather than as neutral lipids, has also

been recognized (Coutteau, Geurden, Camara,

Ber-got & Sorgeloos 1997; Gisbert, Villeneuve,

Zamboni-no-Infante, Quazuguel & Cahu 2005) In addition, in

an attempt to produce preys with high levels of PUFA

to meet essential fatty acid requirements, but that are

not good substrates for energy-generating fatty acid

oxidation systems, the necessity to provide a correct

balance between energy (i.e., saturated and

monoun-saturated fatty acid supply) and essentiality (highly

unsaturated fatty acids; HUFA) should not be

over-looked (Sargent, McEvoy et al 1999) Finally, high

le-vels of highly digestible proteins and peptides, which

are the building blocks for growth, are most probably

required during fast larval growth, although

macro-nutrient requirements have seldom been determined

in marine ¢sh larvae

Advancements in the knowledge of marine ¢sh

lar-vae lipid requirements and in marine biotechnology

and industrial feed processing technologies led to

the development of alternative enrichment products

and additives based in unicellular organisms, such as

yeast, moulds, bacteria and microalgae Some

exam-ples are spray-dried single-cell algal and fungal erotrophic and phototrophic organisms, such asSchizochytrium sp and Crypthecodinium sp., Nano-chloropsis sp., Mortierella alpina or Haematococcus plu-vialis, for instance, that have very high contents ofDHA, EPA, ARA or astaxanthin, respectively, andhigh levels of polar lipids (Harel, Koven, Lein, Bar,Behrens, Stubble¢eld, Zohar & Place 2002; Domin-guez, Ferreira, Coutinho, Fabregas & Otero 2005) In-activated yeast, for instance, can also serve as anadditional source of amino acids and micronutrients,such as nucleic acids, vitamins andb-glucans Thesesingle-cell products have several advantages in rela-tion to emulsi¢ed oil products: PUFA are less exposed

het-to the atmosphere and more protected against tion by the cells, their use minimizes contamination

oxida-of the enrichment media with bacteria that thrive inemulsi¢ed oils and they provide a broader supply ofother natural nutrients, besides lipids (e.g., protein,xanthophylls, vitamins, sterols and other trace ele-ments) (Song, Zhang, Guo, Zhu & Kuang 2007; Yama-saki, Aki, Mori, Yamamoto, Shinozaki, Kawamoto &Onu 2007) Another very important characteristic isthe possibility to combine di¡erent microorganismsproviding rich sources of individual fatty acids,which allows the production of a much larger range

of products capable of meeting the species-speci¢cnutritional requirements of marine ¢sh larvae This

is a major advantage, compared with ¢sh oil-basedproducts, which are limited in terms of the absolutelevels and relative proportions of DHA:EPA:ARA thatcan be supplied Even though high DHA-containing

¢sh oils can be obtained, either from extracting pids from speci¢c tissues (cod liver oil, tuna orbitaloil) or using special extraction procedures (silage,cold acetone), the availability of these products isvery limited and often prohibitively expensive toproduce (Harel et al 2002) Given the present situa-tion of the natural ¢sheries resources, the variablequality, lowering supply and increasing costs of ¢shoil-based products are likely to drive forward the in-dustrial production of the above-mentioned unicel-lular organisms In particular, the production ofheterotrophic algae and fungi in conventional fer-mentors may become an important cost-e¡ectivealternative (Harel et al 2002) Still, emulsion-basedcommercial products using marine ¢sh oils arewidely used but they now tend to include other in-gredients from an extensive list of other stabilizedsupplements, such as free fatty acids, phospholipids,plant and algae extracts, vitamins, minerals, carote-noids and other pigments, antioxidants, proteolyticLive feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

li-r 2009 The Authors

enzymes, immunostimulants and bacteriostatic

additives

Apart from essential fatty acids, whose e¡ects have

been extensively studied and their presence judged to

be critical in marine ¢sh larvae diets, other nutrients,

such as other lipid classes, certain peptides, free

ami-no acids, pigments, sterols, minerals and vitamins,

might be equally important Enrichment procedures

for vitamins A, C and E have been developed since

the 1990s (Merchie 1996; Sorgeloos, Dhert &

Candre-va 2001) but research is required to identify some

of these other dietary components and their

nutri-tional requirement levels, and strategies need to be

devised to include them in an Artemia-based larval

feeding regime (e.g., iodine; Moren, Opstad, Van der

Meeren & Hamre 2006) Liposomes and lipid

mi-crobeads have high potential as delivery systems of

some nutrients, not only of lipid and lipid-soluble

nu-trients but particularly of water-soluble components,

that may not be incorporated e⁄ciently into

emul-sions (Tonheim, Koven & Roennestad 2000; Monroig,

Navarro, Amat, GonzaŁlez, Amat & Hontoria 2003;

Barr & Helland 2007; Nordgreen, Hamre & Langdon

2007)

Rearing techniques forArtemia

Protocols for hatching and enriching brine shrimp

Artemia have been optimized over the years and are

now routinely used in a standardized manner in

commercial hatcheries and research institutes all

over the world A large amount of literature and

prac-tical manuals are widely available (e.g., Lavens &

Sor-geloos 1996; SorSor-geloos et al 2001), and most scienti¢c

research uses standard protocols Therefore, rearing

conditions will only be mentioned brie£y here

Hatching of brine shrimp Artemia is performed in

funnel-shaped containers aerated from the bottom,

at a temperature between 25 and 28 1C, a salinity of

15^35, pH over 8, oxygen levels close to saturation,

cyst densities of not42 g L 1and with strong

illu-mination (around 2000 lx at the water surface) (Van

Stappen 1996b) These conditions will trigger the

start of the Artemia hatching metabolism and have

been optimized to ensure optimal hatching

syn-chrony and e⁄ciency In some hatcheries, bleach is

used to decapsulate the cysts before hatching, to not

only facilitate hatching but also as a prophylactic

measure (to disinfect, i.e., remove bacteria and fungi

that are normally present in cyst shells) In this case,

care should be taken to avoid prolonged exposure to

hypochlorite solution and chlorine then needs to bedeactivated in hydrochloric acid After a period ofaround 24 h, the newly hatched nauplii are separatedfrom unhatched cysts, empty cyst shells (if not decap-sulated) and dead nauplii usually by taking advan-tage of the positive phototactic behaviour of thenauplii, and are thoroughly rinsed with freshwater,preferentially in submerged ¢lters to prevent physicaldamage of the nauplii These are then supplied di-rectly to the larval rearing tanks, when instar I arerequired, or transferred to new clean tanks, forfurther enrichment.When feeding larvae with instar

I nauplii, care should be taken not to supply a surplus

of prey and leave uneaten nauplii for a long time inthe larval tank, as they are consuming their own en-ergy reserves and their level of free amino acids is re-ducing and therefore their nutritional value anddigestibility is quickly decreasing In addition, theyrapidly moult into the second-instar metanauplii,which are larger, faster swimming and more trans-parent (due to the consumption of the brownish or-ange yolk reserves), thus being less accessible preys(Navarro, Amat & Sargent 1991; Merchie 1996; Sorge-loos et al 2001) Storing the newly hatched nauplii at

a temperature below 10 1C in densities of up to 8 lion nauplii L 1, for periods up to 24 h, and distribut-ing them more sparingly and frequently is a way tominimize this problem (Le¤ger,Vanhaecke & Sorgeloos1983; Merchie 1996)

mil-Enrichment is typically conducted at around 28 1C(up to 30 1C) for 24 h in clean, ¢ltered seawater but insome cases manufacturers of speci¢c commercial en-richment products may advise lower temperaturesand enrichment times (e.g., 23^24 1C for 16^20 h).Some of the advantages of reducing temperatureand enrichment time are a reduction in Artemia me-tabolism and DHA catabolism (Navarro et al 1999),thus reducing energy, phospholipid and essentialfatty acid losses, a reduction in the risk of autoxida-tion of HUFA during enrichment (McEvoy, Navarro,Bell & Sargent 1995) and the production of smallerprey sizes, which might be advantageous for somespecies or younger larvae Artemia density is nor-mally 100 000^300 000 nauplii l 1 of water andvigorous aeration is used, to maintain oxygen levelsabove 4 ppm The enrichment product is typicallyblended well with water and added to the enrichment

at a variable dose depending on the manufacturer’sinstructions (often around 0.2^0.3 g L 1for 8^12 h).Usually, two doses are used, with a second dose beingadded to the tank after 8^12 h At the end of the en-richment period, metanauplii are rinsed thoroughly,

sometimes with warm water to help eliminate oily

residues, and are fed to the larvae

Nutritional value ofArtemia

According to Garcia Ortega, Verreth, Coutteau,

Segner, Huisman and Sorgeloos (1998), the proximate

composition (in DW) of newly hatched Artemia is

56.2% protein, 17.0% lipid, 3.6% carbohydrate and

7.6% ash Decapsulated cysts can be used directly as

food for smaller larvae of some freshwater ¢sh and

marine shrimp but its use is much more limited The

composition of decapsulated Artemia cysts is globally

the same as newly hatched nauplii, with about 50^

57% protein, 13^14% lipid, 6^7% carbohydrate and

5^9% ash, but their DW and energy content is on

average 30^40% higher than instar I nauplii On the

other hand, compared with instar I nauplii,

decapsu-lated cysts have a lower ratio of free amino acids to

total protein and of polar lipid and free fatty acids

re-lative to total lipid, vitamin C is present as ascorbic

acid-2 sulphate, which has lower bioavailability and

carotenoids as a less stable form, cis-canthaxanthin

(Van Stappen 1996a; Garcia Ortega et al 1998)

A huge disadvantage of brine shrimp is their

in-herent de¢ciency in essential fatty acids They lack

DHA and have low levels of EPA (even the so-called

marine type) and are richer instead in LNA and to a

lesser extent in linoleic acid (LA;18:2n-6) As already

discussed above, enrichment techniques and

pro-ducts have been developed in order to overcome this

essential fatty acid de¢ciency and are generally quite

e¡ective in boosting the brine shrimp levels of EPA

and ARA However, there still remain di⁄culties in

achieving high levels of DHA and a correct balance

between DHA and EPA, given the natural tendency

of Artemia to retroconvert DHA into EPA (Navarro

et al 1999), leading to a product with a low DHA:EPA

ratio Furthermore, the main PUFA found in Artemia

phospholipids is LNA, followed by EPA, meaning

that very high dietary levels of DHA are required for

the ¢sh larvae to be able to outcompete and replace

the LNA in the ingested phospholipids (in addition to

the high levels found in triacylglycerols), thus

en-abling the larvae to produce DHA-rich phospholipids

required for tissue growth and development (Sargent,

McEnvoy et al., 1999) This has classically been one of

the major drawbacks in the use of Artemia for the

lar-val rearing of most marine ¢sh species Even if ¢sh

larvae have some capacity to reconvert a fraction of

the EPA back into DHA and thus reverse the

undesir-ably high EPA:DHA ratio in the live prey, studies ducted with marine ¢sh indicate that this capacity isextremely limited and certainly insu⁄cient to meetthe high DHA requirements of rapidly growing anddeveloping ¢sh larvae (Sargent, Bell, McEvoy, Tocher

con-& Estevez 1999) Given the extremely important tion of DHA as one of the main components of biolo-gical membranes, particularly in neural tissue, andthe inherent incapability of marine larvae to bio-synthesize it ‘de novo’, DHA nutritional de¢ciencieshave major e¡ects on larval growth and development

func-of neural and visual systems (with major impacts onthe ability of larvae to capture their prey) (Sargent,Bell et al 1999; Sargent, McEvoy et al 1999)

One way to minimize the problem of the lowDHA:EPA ratio in Artemia and to attempt to elevate

it over the 2 or a higher ratio that is advisable for ine ¢sh larvae (Sargent, Bell et al 1999) is to use ‘spe-ciality oils’or marine products with very high levels ofDHA and high DHA:EPA ratios Some of the productsthat have been classically used are tuna orbital oiland either oil extracts or whole-dried or freeze-thawed cells of DHA-rich algae (e.g., Schizochytrium

mar-sp and C cohnii) It should, however, be kept in mindthat the use of these ‘speciality products’ not only hashigh associated costs but they also have limited avail-ability, due to competition with the human consump-tion market, where they are also extensively used(e.g., to supplement infant formulas and used in thehuman food processing industry) Therefore, theyare being commonly used in research, in dose^re-sponse studies (as will be discussed below), but not

as much in routine commercial practices

The protein contents of Artemia are low comparedwith the presumptive requirements of marine ¢shlarvae (Conceicao et al 2003), and with the valuesnormally observed in their natural diet ^ copepods(Table 2; van der Meeren et al 2008) This is particu-larly true as Artemia are normally enriched with lipidemulsions, in an attempt to meet marine ¢sh larvaeDHA requirements This results in a high Artemia li-pid content (mainly neutral lipids) at the expense ofprotein It should be noted that excessive neutral lipid

in Artemia has been shown to a¡ect larval gut tion (see Morais et al 2007 for a review) Moreover,growth is essentially protein deposition, and ¢shlarvae have very high amino acid requirements tosupport fast growth and energy demand (Rnnestad

func-et al 2003; Rnnestad & Conceicao 2005) In tion, the amino acid pro¢le of Artemia protein hasbeen shown to be unbalanced for several larvalspecies (Conceicao, Dersjant, Li & Verreth 1998;Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

addi-r 2009 The Authors

Conceicao et al 2003; Aragao et al 2004; Saavedra

et al 2006, 2007) Such unbalances may cause

higher mortalities (Aragao, Conceicao, Lacuisse,

Yu¤fera & Dinis 2007) or a higher incidence of skeletal

malformations and lower nitrogen utilization

(Saavedra, Pousao-Ferreira,Yu¤fera, Dinis & Conceicao

2009) in ¢sh larvae

Artemia has high contents of canthaxantin,

com-parable to the astaxanthin contents of copepods

(van der Meeren et al 2008) These pigments may

have important roles as antioxidants and sources of

vitamin A in ¢sh larvae nutrition (van der Meeren

et al 2008) Artemia also seem to have high

concen-trations of vitamins C, E, B1 and B2 (van der Meeren

et al 2008) However, iodine, and possibly other

minerals, contents in Artemia may be below the

re-quirements (Moren et al 2006) Iodine limitation has

been identi¢ed as a possible cause of

malpigmenta-tion and other metamorphosis problems in £at¢sh

larvae (Moren et al 2006) due to its precursor role

for thyroid hormones

Microbial aspects inArtemia

The initial content of bacteria in dryArtemia cysts

de-pends on the origin and on the treatment of the cysts

during harvest and packaging Quanti¢cation of the

numbers of aerobic heterotrophic bacteria in

homo-genates of dry Artemia cysts showed that the

num-bers of bacteria associated with the cysts were

equivalent to less than one bacterium per cyst

(Austin 1982) During hatching and enrichment of

Artemia, there is an overload of organic material in

the incubation water, which becomes the substrate

for the proliferation of opportunistic bacteria The

numbers of bacteria increase exponentially and may

reach values as high as 108colony-forming units

(CFU) mL 1in water and 104CFU per Artemia after

enrichment (Villamil, Figueras, Toranzo, Planas &

Novoa 2003) These values may be even higher in the

case of on-grown Artemia (Olsen et al 2000)

Qualita-tive analysis of the Artemia micro£ora indicated that

the largest proportion of bacteria belongs to theVibrio

group, whereVibrio alginolyticus is the dominant

spe-cies (Villamil, Figueras, Toranzo et al 2003) A high

proportion of the bacteria associated with Artemia

metanauplii are haemolytic bacteria (Olsen et al

2000) In the case of newly hatched nauplii, a high

percentage of bacteria are easily removed by rinsing

In contrast, in the case of 24-h enriched Artemia

metanauplii and on-grown Artemia, it is more

di⁄-cult to remove bacteria e⁄ciently by simple rinsing.Metanauplii are able to ¢lter bacteria and accumulatethem in large numbers (Makridis & Vadstein1999) Inthe case of enrichment with Algamac, a commercialspray-dried microalgae, there is even more rapid pro-liferation of bacteria than after incubation with oilemulsions (Ritar, Dunstan, Nelson, Brown, Nichols,Thomas, Smith, Crear & Kolkovski 2004) Severalstrategies have been suggested to limit the presence

of opportunistic bacteria in Artemia used for feeding

of ¢sh larvae, which include the use of probiotic teria that limit the growth of opportunistic bacteria

bac-or have a positive e¡ect on the growth and survival

of Artemia (Verschuere, Rombaut, Huys, Dhont,Sorgeloos & Verstraete 1999; Makridis et al 2000;Gatesoupe 2002; Villamil, Figueras, Planas & Novoa2003), incubation in microalgae (Olsen et al 2000;Makridis et al 2006), or use of disinfectants, such asformaldehyde (Tovar, Zambonino, Cahu, Gatesoupe,VaŁzquez-JuaŁrez & Le¤sel 2002), short-chain acids(Defoirdt, Crab, Wood, Sorgeloos, Verstraete & Bossier2006) and quorum-sensing-disrupting furanones(Defoirdt, Halet, Sorgeloos, Bossier & Verstraete2006; Defoirdt, Boon, Sorgeloos, Verstraete & Bossier2007)

Copepods and other natural zooplanktonMain utilizations of copepods

Copepods and other natural zooplankton organismshave also been used as live feeds, in particular formarine ¢sh larvae In fact, they are the diet for ¢shlarvae of most species in nature Despite signi¢cantprogress in copepod cultivation methods (Payne &Rippingale 2001; Stttrup 2003; Lee, O’Bryen &Marcus 2005), establishing cost-e¡ective protocolsfor mass production is still a challenge Copepodshave mostly been used at a pilot scale or in locationswhere abundant collection of natural (or induced)zooplankton blooms is possible Still, and as could beexpected, the use of copepods as live feeds for marine

¢sh larvae has generally led to considerably better sults in terms of larval performance and quality,when compared with rotifers and/or Artemia (Nss,Germain Henry & Naas 1995; van der Meeren & Naas1997; Stttrup, Shields, Gillespie, Gara, Sargent, Bell,Henderson, Tocher, Sutherland, Nss, Mangor-Jensen, Naas, van der Meeren, Harboe, Sanchez,Sorgeloos, Dhert & Fitzgerald 1998; Shields, Bell, Lui-

re-zi, Gara, Bromage & Sargent 1999; Hamre, Opstad,Espe, Solbakken, Hemre & Pittman 2002; Rajkumar

& Vasagam 2006), in particular for Atlantic halibut

and Atlantic cod larvae Therefore, and whenever

copepods are available, it might be a good strategy

to develop a baseline performance curve using

copepods as live feeds, in particular when developing

larval rearing protocols for ‘new species’ In

situa-tions where copepods are available in limited

amounts, their use as a fraction of the daily ration

for marine ¢sh larvae, in particular during the ¢rst

days of feeding, has also proven to improve larval

growth and survival (Conceicao, van der

Meeren,Ver-reth, Evjen, Houlihan & Fyhn 1997; Toledo, Golez, Doi

& Ohno 1999) This seems particularly interesting for

species requiring very small prey at ¢rst feeding, such

as grouper (Toledo et al 1999; Toledo, Golez & Ohno

2005) and red snapper (Ogle, Lemus, Nicholson,

Barnes & Lotz 2005) Copepod nauplii blooms may

be induced in the rearing tanks or in separate tanks/

ponds It has also been proposed that mass

produc-tion of copepod resting eggs could facilitate

availabil-ity of copepod nauplii for aquaculture (Ns & Bergh

1994; Marcus 2005) However, research is needed on

storage conditions of resting eggs in relation to the

survival and nutritional value of nauplii

Main species of copepods

Free-living copepods belonging to the orders

Calanoi-da, Harpacticoida and Cyclopoida are the most

com-monly used in aquaculture (Stttrup 2003)

Calanoids, e.g., Acartia sp., Eurytemora a⁄nis,

Centro-pages hamatus and Gladioferens imparipes, are by far

the most used for marine ¢sh larvae (see Stttrup

2003 for an overview) However, their maximum

density in culture is an important limitation, and

most Calanoid uses in aquaculture result from

collec-tion of wild populacollec-tions or blooms induced in

con-¢ned areas, such as lagoons or large ponds

Harpacticoids, e.g., Tisbe sp., Euterpina acutifrons,

Tigriopus japonicus and Nitokra sp., are easier to

cul-ture compared with Calanoids Cyclopoids, e.g.,

Apoc-yclops sp and Oithona sp., have been used in only a

few studies (Stttrup 2003) Calanoids are planktonic

in the whole life cycle, while harpacticoids are mostly

benthonic, with a few species having planktonic

nau-pliar stages

Rearing techniques for copepods

Harvest of wild zooplankton has been the most

com-mon source of copepods for ¢sh larviculture

How-ever, extensive and intensive production methodshave also been used (see Stttrup 2003 for a morecomplete overview)

Extensive copepod production can be performed inoutdoor tanks or ponds, and in con¢ned areas such

as lagoons or enclosed fjords Fish larvae may bereared directly in such enclosures/ponds (Stttrup2003) or copepods may be concentrated in the targetsize fractions using ¢ltering devices with di¡erentmesh sizes (van der Meeren & Naas 1997), and laterfed to larvae in tanks, £exible plastic enclosures orponds Extensive production is normally based onmicroalgae blooms induced by an agricultural fertili-zer Nitrogen concentration can be manipulated to fa-vour blooms of certain microalgae types Whennitrogen and oxygen are non-limiting, larger diatomsare normally favoured, leading to higher copepodproductions (Stttrup 2003) Signi¢cant quantities

of marine ¢sh juveniles produced based on extensivecopepod production have been described for grouper

in Philipines (Toledo et al.1999;Toledo et al 2005) andTaiwan (Liao, Su & Chang 2001; Su, Cheng, Chen &

Su 2005), red snapper in the United States (Ogle et al.2005), £ounder in France, cod in Norway and turbot

in Norway and Denmark (Stttrup 2003) However,copepods produced extensively in ponds may causemass mortalities in grouper, through transmission

of viruses (VNN) and parasites (Amyloodinium sp.and gill £ukes) (Su et al 2005)

Intensive mass culture of copepods has been tempted with several species, with varying success,and has resulted in rearing protocols for a number

at-of them (see Stttrup 2003 for further details) A jor problem with copepod intensive production is thelong generation time of most species Species withshorter generation times and with a wider tolerance

ma-to temperature and salinity changes (normally

coast-al species) are preferred for aquaculture (Payne &Rippingale 2001; Stttrup 2003) Culture density isalso a constraint, with many calanoid species su¡er-ing from decreased fecundity due to overcrowding(Miralto, Ianora, Poulet, Romano & Laabir 1996).Thus, harpacticoid copepod species are often men-tioned as the best candidates for mass production.Harpacticoids have the advantages of a high toler-ance to temperature and salinity, ability to feed

on a wide range of live and inert feeds, high fecundity,relatively short life cycles (8^29 days), ability to becultured at high densities (may exceed 100 000 in-dividuals L 1), requirement for surface area ratherthan volume due to their benthonic nature, somehave planktonic naupliar stages and own capabilityLive feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

to clean larvae and copepod rearing tanks surfaces

(Stttrup 2003; Fleeger 2005) In Taiwan, the

cyclo-poid Apocyclops royi has been cultured in the

laboratory fed with microalgae, developing from

nauplius I to copepodite I in 5^6 days with a survival

rate of 89.6% (Liao et al 2001) This makes it a species

with high potential for mass production In contrast,

the European copepod species, Tisbe sp., takes 12^25

days to reach the copepodite I stage with a survival

rate ofo50% (Liao et al 2001)

Intensive production of copepods normally relies

on feeding a combination of at least two species of

mi-croalgae, and ensuring a high n-3 PUFA content

(Stttrup 2003; Knuckey, Semmens, Mayer & Rimmer

2005) However, the calanoid Acartia tonsa has been

successfully reared on defatted rice bran alone (Turk,

Krejci & Yang 1982), and harpacticoids have been

shown to grow on a variety of inert diets (Stttrup

2003; Rhodes & Boyd 2005) Microalgae species fed

to copepods should be chosen depending on the

spe-cies Cell size and concentration is a key factor, and so

is the sinking speed Microalgae such as T suecica,

S costatum and Rhodomonas baltica, which quickly

sediment, are very adequate for benthic harpacticoid

copepods (Stttrup 2003), while I galbana would be

more suitable for calanoids

Aeration is required in copepod intensive

produc-tion, in order to help maintain microalgae in

suspen-sion, promote better algae distribution and avoid

anoxic areas in the tank (Stttrup 2003) Calanoid

culture tanks have a higher requirement for cleaning

by siphoning, because harpacticoids have a

self-cleaning capability Cleaning is essential to avoid

bacterial blooms and ciliate infections, while rotifer

contamination is the most common cause for culture

collapse in large-scale production facilities (see

Stttrup 2003 for a more detailed review on copepod

husbandry techniques)

Nutritional value of copepods

A recent study by van der Meeren et al (2008)

com-prehensively compared the biochemical composition,

as well as the seasonal variation, of copepods

exten-sively produced in a natural enclosure, with that of

rotifers and Artemia enriched with state-of-the-art

technology This study provides detailed information

on major nutrient composition (lipids and amino

acids), as well as micronutrients (pigments and

vita-mins) The biochemical composition of copepods

showed a high stability both between years and

between seasons (van der Meeren et al 2008), and,compared with previous studies, seems to be repre-sentative for the mode for neritic calanoid species.The superior nutritional value of copepods com-pared with Artemia and rotifers has traditionallybeen attributed to their high PUFA, and particularlyHUFA, contents (e.g., Kanazawa 1993; Reitan, Rain-uzzo & Olsen 1994; Bell et al 2003) In fact, despitehaving moderate levels of lipids (6.9^22.5% of DW),copepods have high contents of EPA (8.3^24.6% of to-tal lipid), DHA (13.9^42.3%) and low amounts ofARA (0^2.6%) (van der Meeren et al 2008) Thismeans that copepods have much higher contents ofDHA compared with both enriched rotifers (0.8 fold)

or enriched Artemia (0.3 fold), but only about half theamount of total lipids and ARA (Table 2) In otherwords, in an attempt to meet marine ¢sh larvae DHArequirements, enriched Artemia are loaded with avery high lipid content (mainly neutral lipids) Thesen-3 HUFA contents of copepods also imply EPA/ARAratios usually above 20, and DHA/EPA ratios mostlyabove 2 Such ratios have been shown to be crucialfor optimal marine ¢sh larval performance and qual-ity (Sargent, Bell et al 1999; Bell et al 2003), in parti-cular in £at¢sh, and are very di⁄cult to attain inrotifers and especially in Artemia In fact, copepodsclearly outperform enriched rotifers and enriched Ar-temia in terms of meeting ¢sh larval HUFA require-ments (Bell et al 2003; Rajkumar & Vasagam 2006;van der Meeren et al 2008) The higher contents

in phospholipids (37.9^70.2% of the total lipids) (vander Meeren et al 2008) compared with Artemia (seeTable 2) have also been pointed out as a bene¢t ofcopepods Marine ¢sh larvae have been shown tohave a requirement for dietary phospholipid supply(Geurden 1996; Bell et al 2003; Cahu, Infante &Barbosa 2003) Furthermore, it should be noted thatthe bene¢ts of copepods over rotifers and Artemia arenot just their higher phospholipid content, but thatthe HUFA are predominantly located in the phospho-lipid fraction, while HUFA enrichment in Artemia re-sults in their incorporation largely in the neutral lipidfraction, namely in triacylglycerols (Coutteau &Mourente 1997) This improves the biovailability ofHUFA in copepods compared with cultured liveprey, since HUFA in phospholipids are much morereadily available, digestible and retained in tissuephospholipids, compared with HUFA supplied via aneutral lipid (Izquierdo, Socorro, Arantzamendi &HernaŁndez-Cruz 2000; Gisbert et al 2005)

Copepods also have higher protein and free aminoacid contents compared with Artemia and rotifers

(Table 2; Fyhn, Finn, Helland, Rnnestad & Lomsland

1993; Conceicao et al 1997; Helland, Terjesen & Berg

2003; van der Meeren et al 2008) Free amino acids

and protein constituted between 4.3% and 8.9%,

and 32.7^53.6% of copepod DW respectively (van der

Meeren et al 2008) This means that protein contents

of copepods are on average 0.5 fold higher than those

of Artemia, and copepod nauplii 0.2 fold higher than

rotifers (Table 2) This di¡erence is even higher for

free amino acid contents, with copepods and copepod

nauplii having on average 0.8 and 4.2 fold higher

contents, respectively, compared with Artemia and

rotifers (Table 2) Considering the immature digestive

system of ¢sh larvae, high free amino acid copepod

contents during ¢rst feeding may improve protein

utilization and growth performance (Fyhn et al

1993; Rnnestad et al 2003; Rnnestad & Conceicao

2005) In addition, the superior protein contents of

copepods will contribute to a better use of the high

growth potential of ¢sh larvae (Conceicao et al

2003; Rnnestad et al 2003) Furthermore, the amino

acid pro¢le of copepods seems to better meet larval

grouper amino acid qualitative requirements, and

possibly also those of other species, compared with

rotifers and Artemia (Lacuisse, Conceicao, Lutzki,

Koven,Tandler & Dinis 2005)

Astaxanthin is abundant in copepods (413^

1422mg g 1DW), while Artemia seem to have no

as-taxanthin, but rather comparable amounts of

canthaxantin (Table 2; van der Meeren et al 2008)

These pigments may have important roles as

antioxi-dants and sources of vitamin A in ¢sh larvae

nutri-tion (van der Meeren et al 2008) Copepods, Artemia

and rotifers have high concentrations of vitamins C,

E, B1 and B2 (Table 2; van der Meeren et al 2008)

However, copepods have average iodine contents 109

fold higher compared with Artemia (Table 2; Moren

et al 2006) Iodine limitation has been identi¢ed as a

possible cause of malpigmentation and other

meta-morphosis problems in £at¢sh larvae (Moren et al

2006), due to its precursor role for thyroid hormones

In fact, superior pigmentation as well as survival and

retinal morphology has been documented for halibut

larvae cultured on copepods compared with Artemia

(Shields et al 1999)

Microbial aspects in copepods and other

zooplankton

The numbers of culturable bacteria associated with

copepods are generally much lower than in Artemia,

ranging in values from 5.36 102

nauplius 1ner-Je¡reys, Shields & Birkbeck 2003) The main spe-cies of bacteria associated with marine copepodsbelong to the groups of Vibrio, Pseudomonas and Cy-tophaga (Sochard,Wilson, Austin & Colwell 1979).The use of harvested copepods presents a risk ofintroducing harmful bacteria to larvae and Artemia,

(Ver-as well (Ver-as for humans, such (Ver-as Vibrio cholerae, Vibrioparahaemolyticus,Vibrio vulini¢cus,Vibrio alginolyticusand Aeromonas hydrophila (Rawlings, Ruiz & Colwell2007; Gugliandolo, Irrera, Lentini & Maugeri 2008)

Nutritional studies with live feedsDose^response studies with live feedStudies designed to determine the quantitative re-quirements for speci¢c nutrients require the ability

to analyse dose^response e¡ects However, the treme importance of the relative levels of DHA:EPA:ARA supplied in the diet, and not just theabsolute requirements for essential fatty acids, haslong been recognized These ratios between HUFAhave major implications in physiological mechan-isms in which these fatty acids have a competitive in-teraction, such as in eicosanoid production ormembrane incorporation (Sargent, Bell et al.1999;Sargent, McEnvoy et al., 1999) Originally, theapproach has been to try and simulate the biochem-ical composition of the egg or early larval stages vitel-line reserves or, alternatively, the composition of thelarva’s natural preys (Sargent, Bell et al 1999) How-ever, this approach is quite limited, as the vitelline re-serves are probably only adequate for thedevelopment of the ¢rst early stages of developmentand mimicking the composition of a natural multi-speci¢c zooplankton population is extremely challen-ging In order to address the question of nutrientrequirements, a tight control of the enrichment in in-dividual fatty acids is required, which, as discussedabove, is not an easy task in Artemia However, overthe last few years, advances in the production of spe-ciality oils and the increasing availability of puri¢edsources of fatty acids have enabled the enrichment

ex-of live preys, including rotifers and Artemia, withgraded levels of speci¢c fatty acids, thus enablingdose^response designs to study the essential fattyacid requirements of marine ¢sh larvae (Bransden,Cobcroft, Battaglene, Morehead, Dunstan, Nichols &Kolkovski 2005; Villalta, Este¤vez & Bransden 2005;Villalta, Este¤vez, Bransden & Bell 2005; Lund,Steenfeldt, Banta & Hansen 2008; Villalta, Este¤vez,Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

Bransden & Bell 2008a, b) Studies examining the

biological response of ¢sh larvae to key indicators

of larval performance such as survival, growth,

pigmentation and stress resistance, to feeding live

preys enriched with graded concentrations of the

essential fatty acid of interest or with a gradient of

essential fatty acid ratios (e.g., DHA:EPA or EPA:ARA)

will likely become more common

Live feed labelling

Labelling of live prey has been used to quantify larval

feed intake and/or to characterize digestibility,

ener-getic use or retention of dietary nutrients Both stable

and radioactive isotopes have been used to label both

rotifers and Artemia (e.g., Sorokin & Panov 1966;

Govoni, Peters & Merriner 1982; Boehlert & Yoklavich

1984; Conceicao et al 1998; Conceicao, Skjermo,

Skjak-Braek & Verreth 2001; Morais, Torten, Nixon,

Lutzky, Conceicao, Dinis, Tandler & Koven 2006) A

more comprehensive review of the methods and

ap-plications of tracer studies using live prey can be

found elsewhere (Conceicao, Morais & Rnnestad

2007) The method and tracer molecule that is used

to label the live prey will depend on the objective of

the studies being conducted

Quanti¢cation of food intake has been performed

using either radiolabelled (Sorokin & Panov 1966;

Govoni et al 1982; Boehlert & Yoklavich 1984; Morais

et al 2006), stable isotope-labelled (Conceicao et al

2001) or colour-labelled (Yu¤fera 1987; Parra & Yu¤fera

2000) live preys More recently, small di¡erences in

isotopic signatures (natural abundances of stable

iso-topes) have also been used to study the selectivity of

dietary items by ¢sh larvae (Schlechtriem, Focken &

Becker 2005; Gamboa-Delgado, Canavate, Zerolo &

Le Vay 2008; Jomori, Ducatti, Carneiro & Portella

2008) Artemia incubated in a [U-14C] protein

hydro-lysate has been used to study ontogenetic changes

(Morais, Lacuisse, Conceicao, Dinis & Rnnestad

2004; Engrola 2008) and the impact of the feeding

regime (Engrola 2008) on the protein digestibility of

Senegalese sole early larval stages These studies

es-tablished that Senegalese sole larvae, even at young

stages, have a high capacity for digesting live preys

Co-feeding live feeds with inert diets

The weaning onto commercial feeds usually starts at

the last stages of the larval period, after several weeks

of live prey feeding However, mixed feeding on live

prey plus inert diets during earlier larval stageshas been tested since the 1980s These co-feedingassays have been performed with di¡erent aims: toknow to what degree larvae accept, digest and toler-ate inert diets in order to advance the complete repla-cement of live prey; to ¢nd a way to deliver speci¢ccompounds into the larval gut; and mainly to per-form an early weaning to reduce dependence onlive prey

Di¡erent commercial and experimental microdietshave been tested in di¡erent relative proportions withrotifers and Artemia (Kanazawa, Koshio & Teshima1989; Holt1993; Hart & Purser1996; Rosenlund, Stoss

& Talbot 1997; Canavate & FernaŁndez-D|¤az 1999;Alves, Cerqueira & Brown 2006; Chang, Liang,Wang,Chen, Zhang & Liu 2006; Aristizabal & Suarez 2007;Fletcher, Roy, Davie, Taylor, Robertson & Migaud2007; Engrola 2008) In general, these studies showthat ¢sh larvae of di¡erent species grow very well inco-feeding when the live prey substitution level is notexcessive More rarely, a high substitution ratio(Kanazawa et al 1989; Yu¤fera, Kolkovski, FernaŁndez-D|¤az, Rinchard, Lee & Dabrowski 2003) or even thecomplete substitution (Fontagne¤, Robin, Corraze &Bergot 2000) also yielded good growth results Anearly co-feeding period seems to prepare the gut foraccepting and processing inert diets, allowing earlierweaning and with better growth performances thanwhen weaning starts at the end of the larval stage(Rosenlund et al 1997; Canavate & FernaŁndez-D|¤az1999; Curnow, King, Bosmans & Kolkovski 2006; En-grola 2008) Fish larvae clearly prefer live prey toinert diets (FernaŁndez-D|¤az et al 1994) and in somespecies the acceptance of inert diets is worse after along feeding period on live prey exclusively In addi-tion, the co-feeding regime may contribute to correctpotential nutrient de¢ciencies

ConclusionsLive feeds are still the main feed item in commerciallarviculture, despite their nutritional composition of-ten being sub-optimal Further improvements in in-ert microdiet technology and formulation will likelylead to a progressive substitution of live feeds How-ever, this substitution will be gradual and is mostlikely far from being complete for many species, atleast for the ¢rst days of feeding Co-feeding of smallamounts of live feeds with high-quality inert micro-diets will likely lead to major improvements in larvalperformance Such feeding strategies may take ad-

vantage of the strong points of both live feeds

(stimu-lation of feeding behaviour) and inert diets

(opti-mized nutritional composition)

Acknowledgments

This review was partially supported by projects:

POCI/MAR/61623/2004 ^ SAARGO, ¢nanced by

pro-gram POCI 2010 (FCT, Portugal), which is

co-¢-nanced by FEDER; and project P06-AGR-01697

funded by Consejer|¤a Innovacio¤n, Ciencia y Empresa

^ Junta de Andaluc|¤a (Spain)1FEDER

References

Alves T.T., CerqueiraV.R & Brown J.A (2006) Early weaning

of fat snook (Centropomus parallelus Poey 1864) larvae.

Aquaculture 253, 334^342.

Aragao C., Conceicao L.E.C., Dinis M.T & Fyhn H.J (2004)

Amino acid pools of rotifers and Artemia under di¡erent

conditions: nutritional implications for ¢sh larvae

Aqua-culture 234, 429^445.

Aragao C., Conceicao L.E.C., Lacuisse M.,Yu¤fera M & Dinis

M.T (2007) Do dietary amino acid pro¢les a¡ect

perfor-mance of larval gilthead seabream? Aquatic Living

Re-sources 20, 155^161.

Aristizabal E.O & Suarez J (2007) E⁄ciency of co-feeding

red porgy (Pagrus pagrus L.) larvae withy live and

com-pound diet Revista de biolog|¤a marina y oceanograf|¤a 41,

203^208.

Austin B (1982) Taxonomy of bacteria isolated from a

coast-al marine ¢sh-rearing unit Journcoast-al of Applied Bacteriology

53, 253^268.

Austin B & Day J.G (1990) Inhibition of prawn pathogenic

Vibrio spp by a commercial spray-dried preparation of

Tetraselmis suecica Aquaculture 90, 389^392.

Austin B., Baudet E & Stobie M (1992) Inhibition of

bacter-ial ¢sh pathogens byTetraselmis suecica Journal of Fish

Dis-eases 15, 55^61.

Baer A., Langdon C., Mills S., Schulz C & Hamre K (2008)

Particle size preference, gut ¢lling and evacuation rates

of the rotifer Brachionus ‘‘Cayman’’ using polystyrene

la-tex beads Aquaculture 282,75^82.

Barr Y & Helland S (2007) A simple method for

mass-pro-duction of liposomes, in particular large liposomes,

suita-ble for delivery of free amino acids to ¢lter feeding

zooplankton Journal of Liposome Research 17,79^88.

Baxevanis A.D., Kappas I & Abatzopoulos T.J (2006)

Mole-cular phylogenetics and asexuality in the brine shrimp

Artemia Molecular Phylogenetics and Evolution 40, 724^

738.

Becker W (2004) Microalgae for aquaculture The

nutri-tional value of microalgae for aquaculture in Handbook

of Microalgal Culture Biotechnology and Applied Phycology

(ed by A Richmond), pp 380^391 Blackwell Science, Ames, IA, USA.

Bell J.G., McEvoy L.A., Estevez A., Shields R.J & Sargent J.R (2003) Optimising lipid nutrition in ¢rst-feeding £at¢sh larvae Aquaculture 227, 211^220.

Ben-Amotz A., Fishler R & Schneller A (1987) Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids Marine Biology

95, 31^36.

Bengtson D.A (2003) Status of marine aquaculture in tion to live prey: past, present and future in Live Feeds in Marine Aquaculture (ed by J.G Strttrup & L.A McEvoy),

rela-pp 1^16 Blackwell publishing, Oxford, UK.

Boehlert G.W & Yoklavich M.M (1984) Carbon assimilation

as a function of ingestion rate in larval Paci¢c herring, Clupea harengus pallasi Valenciennes Journal of Experi- mental Marine Biology and Ecology 79, 251^262 Boraas M.E (1983) Population dynamics of food-limited roti- fers in two-stage chemostat culture Limnology and Ocea- nography 28, 546^563.

Bransden M.P., Cobcroft J.M., Battaglene S.C., Morehead D.T., Dunstan G.A., Nichols P.D & Kolkovski S (2005) Dietary 22:6n-3 alters gut and liver structure and beha- viour in larval striped trumpeter (Latris lineata) Aquacul- ture 248, 275^285.

Brown M.R (1991) The amino acid and sugar composition

of 16 species of microalgae used in mariculture Journal of Experimental Marine Biology and Ecology 145, 79^99.

Cahu C & Infante J.Z (2001) Substitution of live food by mulated diets in marine ¢sh larvae Aquaculture 200, 161^180.

for-Cahu C.L., Infante J.L.Z & Barbosa V (2003) E¡ect of dietary phospholipid level and phospholipid: neutral lipid value

on the development of sea bass (Dicentrarchus labrax) vae fed a compound diet British Journal of Nutrition 90, 21^28.

lar-Canavate J.P & FernaŁndez-D|¤az C (1999) In£uence of feeding larvae with live and inert diets on weaning the sole Solea senegalensis onto commercial dry feeds Aqua- culture 174, 255^263.

co-Carvalho A.P., SaŁ R., Oliva-Teles A & Bergot P (2004) lity and peptide pro¢le a¡ect the utilization of dietary pro- tein by common carp (Cyprinus carpio) during early larval stages Aquaculture 234, 319^333.

Solubi-Chang Q., Liang M.Q., Wang J.L., Chen S.Q., Zhang X.M & Liu X.D (2006) In£uence of larval co-feeding with live and inert diets on weaning the tongue sole Cynoglossus semilaevis Aquaculture Nutrition 12, 135^139.

Ciros-Pe¤rez J., Go¤mez A & Serra M (2001) On the taxonomy

of three sympatric sibling species of the Brachionus tilis (Rotifera) complex from Spain, with the description of

plica-B ibericus n sp Journal of Plankton Research 23, 1311^ 1328.

Conceicao L.E.C., van der Meeren T., Verreth J.A.J., Evjen M.S., Houlihan D.F & Fyhn H.J (1997) Amino acid meta- Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

bolism and protein turnover in larval turbot

(Scophthal-mus maxi(Scophthal-mus) fed natural zooplankton orArtemia Marine

Biology 129, 255^265.

Conceicao L.E.C., Dersjant-Li Y & Verreth J.A.J (1998) Cost of

growth in larval and juvenile African cat¢sh (Clarias

gariepinus) in relation to growth rate, food intake and

oxy-gen consumption Aquaculture 161, 95–106.

Conceicao L.E.C., Skjermo J., Skjak-Braek G & Verreth J.A.J.

(2001) E¡ect of an immunostimulating alginate on

pro-tein turnover of turbot (Scophthalmus maximus L.) larvae.

Fish Physiology and Biochemistry 24, 207^212.

Conceicao L.E.C., Grasdalen H & Ronnestad I (2003) Amino

acid requirements of ¢sh larvae and post-larvae: new

tools and recent ¢ndings Aquaculture 227, 221^232.

Conceicao L.E.C., Morais S & Rnnestad I (2007) Tracers in

¢sh larvae nutrition: a review of methods and

applica-tions Aquaculture 267, 62^75.

Coutteau P & Sorgeloos P (1992) The use of algal substitutes

and the requirement for live algae in the hatchery and

nursery rearing of bivalve molluscs: an international

sur-vey Journal of Shell¢sh Research 11, 467^476.

Coutteau P & Mourente G (1997) Lipid classes and their

content of n-3 highly unsaturated fatty acids (HUFA) in

Artemia franciscana after hatching, HUFA-enrichment

and subsequent starvation Marine Biology 130, 81^91.

Coutteau P., Geurden I., Camara M.R., Bergot P & Sorgeloos

P (1997) Review on the dietary e¡ects of phospholipids

in ¢sh and crustacean larviculture Aquaculture 155,

149^164.

Curnow J., King J., Bosmans J & Kolkovski S (2006) The

ef-fect of reduced Artemia and rotifer use facilitated by a new

microdiet in the rearing of barramundi Lates calcarifer

(BLOCH) larvae Aquaculture 257, 204^213.

Curnow J., King J., Partridge G & Kolkovski S (2006) E¡ects

of two commercial microdiets on growth and survival of

barramundi (Lates calcarifer Bloch) larvae within various

early weaning protocols Aquaculture Nutrition 12, 247^

255.

De Swaarf M.E., Pronk J.T & Sijtsma L (2003) Fed-batch

cul-tivation of the docosahexaenoic-acid-producing marine

alga Crypthecodinium cohnii on ethanol Applied

Micro-biology and Biotechnology 61, 40^43.

Defoirdt T., Crab R., Wood T.K., Sorgeloos P., Verstraete W &

Bossier P (2006) Quorum sensing-disrupting brominated

furanones protect the gnotobiotic brine shrimp Artemia

franciscana from pathogenic Vibrio harveyi, Vibrio

camp-bellii, andVibrio parahaemolyticus isolates Applied and

En-vironmental Microbiology 72, 6419^6423.

Defoirdt T., Halet D., Sorgeloos P., Bossier P & Verstraete W.

(2006) Short-chain fatty acids protect gnotobiotic Artemia

franciscana from pathogenic Vibrio campbellii

Aquacul-ture 261, 804^808.

Defoirdt T., Boon N., Sorgeloos P., Verstraete W & Bossier P.

(2007) Alternatives to antibiotics to control bacterial

in-fections: luminescent Vibriosis in aquaculture as an

ex-ample Trends in Biotechnology 25, 472^479.

Dhert P., Rombaut G., Suantika G & Sorgeloos P (2001) vancement of rotifer culture and manipulation techni- ques in Europe Aquaculture 200, 129^146.

Ad-Dominguez A., Ferreira M., Coutinho P., Fabregas J & Otero

A (2005) Delivery of astaxanthin from Haematococcus pluvialis to the aquaculture food chain Aquaculture 250, 424^430.

Dooms S., Papakostas S., Ho¡man S., Delbare D., Dierkens K.,Triantafyllidis A., De Wolf T.,Vadstein O., Abatzopoulos T.J., Sorgeloos P & Bossier P (2007) Denaturing gradient gel electrophoresis (DGGE) as a tool for the characterisa- tion of Brachionus sp strains Aquaculture 262, 29^40 Douillet P.A (2000) Bacterial additives that consistently en- hance rotifer growth under synxenic culture conditions.

1 Evaluation of commercial products and pure isolates Aquaculture 182, 249^260.

Droop M.R & Scott J.M (1978) Steady-state energetics of a planktonic herbivore Journal of the Marine Biological As- sociation of the United Kingdom 58,749^772.

Du¡ D.C.B & Bruce D.L (1966) The antibacterial activity of marine planktonic algae CanadianJournal of Microbiology

12, 877^884.

Engrola S (2008) Improving growth performance of Senegalese sole postlarvae PhD Thesis University of Algarve, Faro, 167pp.

Faulk C.K & Holt G.J (2005) Advances in rearing cobia chycentron canadum larvae in recirculating aquaculture systems: live prey enrichment and greenwater culture Aquaculture 249, 231^243.

Ra-FernaŁndez-D|¤az C., Pascual E & Yu¤fera M (1994) Feeding havior and prey size selection of gilthead seabream, Sparus aurata, larvae fed on inert and live food Marine Biology 118, 323^328.

be-FernaŁndez-Reiriz M.J & Labarta U (1996) Lipid classes and fatty acid composition of rotifer (Brachionus plicatilis) fed two algal diets Hydrobiologia 330,73^79.

FernaŁndez-Reiriz M.J., Labarta U & Ferreiro M.J (1993) fects of commercial enrichment diets on the nutritional value of the rotifer (Brachionus plicatilis) Aquaculture

Ef-112, 195^206.

Fernandez R.G (2001) Artemia bioencapsulation I E¡ect of particle sizes on the ¢ltering behavior of Artemia francis- cana Journal of Crustacean Biology 21, 435^442 Fleeger J.W (2005) The potential to mass-culture harpacti- coid copepods for use as food for larval ¢sh in Copepods

in Aquaculture (ed by C.-S Lee, P.J O’Bryen & N.H cus), pp 11^24 Blackwell Publishing, Oxford, UK Fletcher R.C., Roy W., Davie A.,Taylor J., Robertson D & Mi- gaud H (2007) Evaluation of new microparticulate diets for early weaning of Atlantic cod (Gadus morhua): implica- tions on larval performances and tank hygiene Aquacul- ture 263, 35^51.

Mar-Fogg G.E (1975) Algal Cultures and Phytoplankton Ecology University of Wisconsin Press, Madison,WI, USA, 175pp Fontagne¤ S., Robin J., Corraze G & Bergot P (2000) Growth and survival of European sea bass (Dicentrarchus labrax)

larvae fed from ¢rst feeding on compound diets

contain-ing mediumçchain triacylglycerols Aquaculture 190,

261^271.

Fontaneto D., Giordani I & Serra M (2007) Disentangling

the morphological stasis in two rotifer species of the

Brachionus plicatilis species complex Hydrobiologia 583,

297^307.

Fu Y., Hirayama K & Natsukari Y (1991) Genetic divergence

between S and L type strains of the rotifer Brachionus

pli-catilis O.F Mˇller Journal of Experimental Marine Biology

and Ecology 151, 43^56.

FuY., Ada A.,Yamashita T.,YashidaY & Hino A (1997)

Devel-opment of a continuous culture system for stable mass

production of rhe rotifer marine Brachionus

Hydrobiolo-gia 358, 141^151.

Fukami K., Nishijima T & Ishida Y (1997) Stimulative and

inhibitory e¡ects of bacteria on the growth of microalgae.

Hydrobiologia 358, 185^191.

Fukusho K & Okaushi M (1983) Sympatry in natural

distri-bution of the two strains of a rotifer, Brachionus plicatilis.

Bulletin of National Research Institure of Aquaculture 4,

135^138.

Fyhn H.J., Finn R., Helland S., Rnnestad I & Lomsland E.R.

(1993) Nutritional value of phyto- and zooplankton as live

food for marine ¢sh larvae In: Fish Farming Technology

(ed by H Reinertsen, L.A Dahle, L Jørgensen & K.

Tvinnerheim), pp 121–126 Balkema, Rotterdam, the

Netherlands.

Gamboa-Delgado J., Canavate J.P., Zerolo R & Le Vay L.

(2008) Natural carbon stable isotope ratios as indicators

of the relative contribution of live and inert diets to

growth in larval Senegalese sole (Solea senegalensis).

Aquaculture 280, 190^197.

Garcia Ortega A.,Verreth J.A.J., Coutteau P., Segner H.,

Huis-man E.A & Sorgeloos P (1998) Biochemical and

enzy-matic characterization of decapsulated cysts and nauplii

of the brine shrimp Artemia at di¡erent developmental

stages Aquaculture 161, 501^514.

Gatesoupe F.J (2002) Probiotic and formaldehyde

treat-ments of Artemia nauplii as food for larval pollack,

Polla-chius pollaPolla-chius Aquaculture 212, 347^360.

Gatesoupe F.J & Luquet P (1981) Practical diet for mass

cul-ture of the rotifer Brachionus plicatilis: application to

lar-val rearing of sea bass, Dicentrarchus labrax Aquaculture

22, 149^163.

Gatesoupe F.J & Robin J.H (1981) Commercial single-cell

proteins either as sole source or in formulated diets for

in-tensive and continuous production of rotifer Brachionus

plicatilis Aquaculture 25, 1^15.

Gatesoupe F.J., Arakawa T & WatanabeT (1989) The e¡ect of

bacterial additives on the production rate and dietary

va-lue of rotifers as food for Japanese £ounder, Paralicthys

oli-vaceus Aquaculture 83, 39^44.

Geurden I (1996) The role of phospholipids in diets of larval and

postlarval ¢sh PhD Thesis Universiteit Gent,

Landbouw-kunde, Belgium, 239pp.

Gime¤nez G., KotzamanisY., Hontoria F., Estevez A & Gisbert

E (2007) Modelling retinoid content in live prey: a tool for evaluating the nutritional requirement and development studies in ¢sh larvae Aquaculture 267,76^82.

Gisbert E.,Villeneuve L., Zambonino-Infante J.L., Quazuguel

P & Cahu C.L (2005) Dietary phospholipids are more cient than neutral lipids for long-chain polyunsaturated fatty acid supply in European sea bass Dicentrarchus lab- rax larval development Lipids 40, 609^618.

e⁄-Go¤mez A & Serra M (1995) Behavioural reproductive tion among sympatric strains of Brachionus plicatilis Mul- ler 1786: insights into status of this taxonomic species Hydrobiologia 313/314, 111^119.

isola-Go¤mez A., Serra M., Carvalho G.R & Lunt D.H (2002) ciantion in anvcient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis (Ro- tifera) Evolution 56, 1431^1444.

Spe-Govoni J.J., Peters D.S & Merriner J.V (1982) Carbon tion during larval development of the marine teleost Leiostomus xanthurus Lacepede Journal of Experimental Marine Biology and Ecology 64, 287^299.

assimila-Govoni J.J., Boehlert G.W & Watanabe Y (1986) The ogy of digestion in ¢sh larvae Environmental Biology of Fishes 16, 59^77.

physiol-Gugliandolo C., Irrera G.P., Lentini V & Maugeri T.L (2008) Pathogenic Vibrio, Aeromonas and Arcobacter spp asso- ciated with copepods in the Straits of Messina (Italy) Mar- ine Pollution Bulletin 56, 600^606.

Hamre K., Opstad I., Espe M., Solbakken J., Hemre G.-I & Pittman K (2002) Nutrient composition and metamor- phosis success of Atlantic halibut (Hippoglossus hippoglos- sus, L.) larvae fed natural zooplankton or Artemia Aquaculture Nutrition 8, 139^148.

Hamre K., Mollan T.A., Sle  & Erstad B (2008) Rotifers enriched with iodine and selenium increase survival in Atlantic (Gadus morhua) cod larvae Aquaculture 284, 190–195.

Hamre K., Srivastava A., Rnnestad I., Mangor-Jensen A & Stoss J (2008) Several micronutrients in the rotifer Bra- chionus sp may not ful¢l the nutritional requirements of marine ¢sh larvae Aquaculture Nutrition 14, 51^60 Hamza N., Mhetli M & Kestemont P (2007) E¡ects of wean- ing age and diets on ontogeny of digestive activities and structures of pikeperch (Sander lucioperca) larvae Fish Physiology and Biochemistry 33, 121^133.

Harel M., KovenW., Lein I., BarY., Behrens P., Stubble¢eld J., ZoharY & Place A.R (2002) Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using sin- gle cell heterotrophs Aquaculture 213, 347^362 Hart P.R & Purser G.J (1996) Weaning of hatchery-reared greenback £ounder (Rhombosolea tapirina Gˇnther) from live to arti¢cial diets: e¡ects of age and duration of the changeover period Aquaculture 145, 171^181.

Heasman M., Diemar J., O’Connor W., Sushames T & Foulkes

L (2000) Development of extended shelf-life micro-algae Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

concentrate diets harvested by centrifugation for bivalve

molluscs-a summary Aquaculture Research 31, 637^659.

Helland S.,Terjesen B.F & Berg L (2003) Free amino acid and

protein content in the planktonic copepod Temora

longi-cornis compared to Artemia franciscana Aquaculture

215, 213^228.

Hirata H (1974) An attempt to apply an experimental

micro-cosm for the mass culture of marine rotifer, Brachionus

plicatilis Mˇller Memoirs of the Faculty of Fisheries 32,

163–172.

Hirata H & Mori Y (1967) Culture of the rotifer Brachionus

plicatilis fed on baker’s yeast Saibai-Gyogyo 5, 36^40.

Hirayama K & Kusano T (1972) Fundamental studies on

physiology of the rotifer for its mass culture II In£uence

of water temperature on population growth of rotifer

Bul-letin of the Japanese Society of Scienti¢c Fisheries 38, 1357^

1363.

Hirayama K & Nakamura K (1976) Fundamental studies on

physiology of rotifers in mass culture 5 Dry Chlorella

powder as a food for rotifers Aquaculture 8, 301^307.

Hirayama K.,Watanabe K & Kusano T (1973) Fundamental

studies on physiology of the rotifer for its mass culture III.

In£uence of phytoplankton density on population

growth Bulletin of the Japanese Society of Scienti¢c

Fish-eries 39, 1123^1127.

Hirayama K., Takaga K & Kimura H (1979) Nutritional

ef-fect of eight species of marine phytoplankton on

popula-tion growth of the rotifer Brachionus plicatilis Bulletin of

the Japanese Society of Scienti¢c Fisheries 45, 11^16.

Holt G.J (1993) Feeding larval red drum on microparticulate

diets in a closed recirculating water system Journal of the

World Aquaculture Society 24, 225^230.

Hou L., Bi X., Zou X., He C.,Yang L., Qu R & Liu Z.W (2006)

Molecular systematics of bisexual Artemia populations.

Aquaculture Research 37, 671^680.

Ii H., Hirata T., Matsuo H., Nishikawa M & Tase N (1997)

Surface water chemistry, particularly concentrations of

NO3- and DO and delta N-15 values, near a tea plantation

in Kyushu, Japan Journal of Hydrology 202, 341^352.

Ito T (1960) On the culture of the mixohaline rotifer

Bra-chionus plicatilis O.F Muller in the sea water Department

of the Faculty of Fisheries, Mie Prefecture University 33,

708^740.

Izquierdo M.S., Socorro J., Arantzamendi L &

HernaŁndez-Cruz C.M (2000) Recent advances in lipid nutrition in

¢sh larvae Fish Physiology and Biochemistry 22, 97^107.

James C.M & Abu Rezeq T (1989) Intensive rotifer cultures

using chemostats Hydrobiologia 186/187, 43^50.

James C.M., Dias P & Salman A.E (1987) The use of marine

yeast (Candida sp.) and baker’s yeast (Saccharomyces

cerevisiae) in combination with Chlorella sp for mass

cul-ture of rotifer Brachionus plicatilis Hydrobiologia 147,

263^268.

Jomori R.K., Ducatti C., Carneiro D.J & Portella M.C (2008)

Stable carbon (13C) and nitrogen (d15N) isotopes as

nat-ural indicators of live and dry food in Piaractus

mesopota-micus (Holmberg,1887) larval tissue Aquaculture Research

39, 370^381.

Kanazawa A (1993) Importance of dietary docosahexaenoic acid on growth and survival of ¢sh larvae In Fin¢sh Hatchery in Asia Proceedings of Fin¢sh Hatchery in Asia ’

91 (ed by C.S Lee, M.S Su & I.C Liao), pp 87–95 Tungkang Marine Laboratory, Taiwan.

Kanazawa A., Koshio S & Teshima S.-I (1989) Growth and survival of larval red sea bream Pagrus major and Japanese £ounder Paralichthys olivaceus fed microbound diets Journal of theWorld Aquaculture Society 20, 31^37 Klaoudatos S.D., Iakovopoulos G & Klaoudatos D.S (2004) Pagellus erythrinus (Common Pandora): a pro- mising candidate species for enlarging the diversity of aquaculture production Aquaculture International 12, 299^320.

Knauer J & Southgate P.C (1999) A review of the nutritional requirements of bivalves and the development of alterna- tive and arti¢cial diets for bivalve aquaculture Reviews in Fisheries Science 7, 241^280.

Knuckey R.M., Semmens G.L., Mayer R.J & Rimmer M.A (2005) Development of an optimal microalgal diet for the culture of the calanoid copepod Acartia sinjiensis: e¡ect of algal species and feed concentration on copepod develop- ment Aquaculture 249, 339^351.

Knuckey R.M., Brown M.R., Robert R & Frampton D.M.F (2006) Production of microalgae concentrates by £occu- lation and their assessement as aquaculture feeds Aqua- culture Engineering 35, 300^313.

Kokou F., Ferreira T.,Tsigenopoulos C.S., Makridis P., las G., Magoulas A & Divanach P (2007) Identi¢cation of bacteria growing in association with batch cultures of the microalgae Chlorella minutissima In 8th International Marine Biotechnology Conference, pp 105 Eilat, Israel Korstad J., Olsen Y & Vadstein O (1989) Life history charac- teristics of Brachionus plicatilis (Rotifera) fed di¡erent al- gae Hydrobiologia 186, 43^50.

Kotou-KostopoulouV & Vadstein O (2007) Growth performance of the rotifers Brachionus plicatilis, B.‘Nevada’and B.‘Cayman’ under di¡erent food concentrations Aquaculture 273, 449^458.

Koven W., Kolkovski S., Hadas E., Gamsiz K & Tandler A (2001) Advances in the development of microdiets for gilt- head seabream, Sparus aurata: a review Aquaculture 194, 107^121.

Lacuisse M., Conceicao L., Lutzki S., Koven W.,Tandler A & Dinis M.T (2005) Do copepods meet the a requirements of the white grouper larvae better than rotifers and Artemia?

In LarviŁ05-Fish & Shell¢sh Larviculture Symposium, pp 268^271, Oostende, Belgium.

Langdon C & nal E (1999) Replacement of living gae with spray-dried diets for the marine mussel Mytilus galloprovincialis Aquaculture 180, 283^294.

microal-Lavens P & Sorgeloos P (1996) Manual on the production and use of live food for aquaculture FAO Technical Papers 361 FAO, Rome, Italy, 295pp.

Lavens P & Sorgeloos P (2000) The history, present status

and prospects of the availability of Artemia cysts for

aqua-culture Aquaculture 181, 397^403.

Lazo J.P., Dinis M.T., Holt J.G., Faulk C & Arnold C.R (2000)

Co-feeding microparticulate diets with algae: towards

eliminating the need of zooplankton at ¢rst feeding

in red drum (Sciaenops ocellatus) Aquaculture 188,

339^351.

Lee C.-S., O’Bryen P.J & Marcus N.H (2005) Copepods in

Aquaculture Blackwell Publishing, Oxford, UK, 269pp.

Le¤ger P., Vanhaecke P & Sorgeloos P (1983) International

Study on Artemia XXIV Cold storage of live Artemia

nauplii from various geographical sources: potentials

and limits in aquaculture Aquaculture Engineering 2, 69^

78.

Liao I.C., Su H.M & Chang E.Y (2001) Techniques in ¢n¢sh

larviculture in Taiwan Aquaculture 200, 1^31.

Lubzens E (1987) Raising rotifers for use in aquaculture.

Hydrobiologia 147, 245^255.

Lubzens E & Zmora O (2003) Production and nutritional

va-lue of rotifers in Live Feeds in Marine Aquaculture (ed by

L.A McEvoy), pp 17^64 Blackwell publishing, Oxford,

UK.

Lubzens E.,Tandler A & Minko¡ G (1989) Rotifers as food in

aquaculture Hydrobiologia 186/187, 387^400.

Lund I., Steenfeldt S.J., Banta G & Hansen B.W (2008) The

in£uence of dietary concentrations of arachidonic acid

and eicosapentaenoic acid at various stages of larval

on-togeny on eye migration, pigmentation and prostaglandin

content of common sole larvae (Solea solea L.) Aquaculture

276, 143^153.

Makridis P (2000) Methods for the microbial control of live

food used for the rearing of marine ¢sh larvae Dr scient.

Thesis Norwegian University of Science and Technology,

Greece, pp 1^19.

Makridis P & Vadstein O (1999) Food size selectivity of

Arte-mia franciscana at three developmental stages Journal of

Plankton Research 21, 2191^2201.

Makridis P & Olsen Y (1999) Protein depletion of the rotifer

Brachionus plicatilis during starvation Aquaculture 174,

343^353.

Makridis P., Fjellheim J.A., Skjermo J & Vadstein O (2000)

Control of the bacterial £ora of Brachionus plicatilis and

Artemia franciscana by incubation in bacterial

suspen-sions Aquaculture 185, 207^218.

Makridis P., Alves Costa R & Dinis M.T (2006) Microbial

conditions and antimicrobial activity in cultures of two

microalgae species Tetraselmis chuii and Chlorella

minutis-sima, and e¡ect on bacterial load of enriched Artemia

me-tanauplii Aquaculture 255,76^81.

Makridis P., Martins S., Reis J & Dinis M.T (2008) Use of

probiotic bacteria in the rearing of Senegalese sole

(Solea senegalensis) larvae Aquaculture Research 39, 627^

634.

Marcus N.H (2005) Calanoid copepods, resting eggs, and

aquaculture in Copepods in Aquaculture (ed by C.-S Lee,

P.J O’Bryen & N.H Marcus), pp.3^9 Blackwell Publishing, Oxford, UK.

Marshall J.A., de Salas M., Oda T & Hallegraef G (2005) Superoxide production by marine microalgaeçI Survey

of 37 species from 6 classes Marine Biology147,533^540 McEvoy L.A., Navarro J.C., Bell J.G & Sargent J.R (1995) Autoxidation of oil emulsions during the Artemia enrich- ment process Aquaculture 134, 101^112.

McIntosh D., Ji B., Forward B.S., PuvanendranV., Boyce D & Ritchie R (2008) Culture-independent characterization

of the bacterial populations associated with cod (Gadus morhua L.) and live feed at an experimental hatchery facil- ity using denaturing gradient gel electrophoresis Aqua- culture 275, 42^50.

Merchie G (1996) Use of nauplii and meta-nauplii In: ual on the production and use of live food for aquaculture FAO Fisheries Technical Paper 361 (ed by P Lavens & P Sorge- loos), pp 137^163 FAO, Rome, Italy.

Man-Mills S., Lunt D.H & Go¤mez A (2007) Global isolation by tance despite strong regional phylogeography in a small metazoan BMC Evolucionary Biology 7, 225.

dis-Miracle M.R & Serra M (1989) Salinity and temperature

in-£uence in rotifer life history characteristics Hydrobiologia 186/187, 81^102.

Miralto A., Ianora A., Poulet S.A., Romano G & Laabir M (1996) Is fecundity modi¢ed by crowding in the copepod Centropages typicus Journal of Plankton Research 18, 1033^ 1040.

Mo¡att N.M (1981) Survival and growth of northern

ancho-vy larvae on low zooplankton densities as a¡ected by the presence of a Chlorella bloom Rapport Procedures Re¤union

du Conseil internationale de Exploration de la Mer 178, 475^ 480.

Monroig O., Navarro J.C., Amat I., GonzaŁlez P., Amat F & Hontoria F (2003) Enrichment of Artemia nauplii in PUFA, phospholipids, and water-soluble nutrients using liposomes Aquaculture International 11, 151^161 Morais S., Lacuisse M., Conceicao L.E.C., Dinis M.T & Rn- nestad I (2004) Ontogeny of the digestive capacity of (So- lea senegalensis), with respect to and metabolism of amino acids from Senegalese sole digestion, absorption Artemia Marine Biology 145, 243^250.

Morais S., Torten M., Nixon O., Lutzky S., Conceicao L.E.C., Dinis M.T.,Tandler A & Koven W (2006) Food intake and absorption are a¡ected by dietary lipid level and lipid source in seabream (Sparus aurata L.) larvae Journal of Ex- perimental Marine Biology and Ecology 331, 51^63 Morais S., Conceicao L.E.C., Rnnestad I., Koven W., Cahu C., Infante J.L.Z & Dinis M.T (2007) Dietary neutral lipid level and source in marine ¢sh larvae: e¡ects on digestive physiology and food intake Aquaculture 268, 106^122 Moren M., Opstad I., Van der Meeren T & Hamre K (2006) Iodine enrichment of Artemia and enhanced levels of io- dine in Atlantic halibut larvae (Hippoglossus hippoglossus L.) fed the enriched Artemia Aquaculture Nutrition 12, 97^ 102.

Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

Muller-Fuega A., Moal J & Kaasa R (2004) Microalgae for

aquaculture The current global situation and future

trends in Microalgal Culture Biotechnology and Applied

Phycology (ed by A Richmond), pp 352^364 Blackwell

Science, Pondicherry, India.

Munro P.D., McLean H.A., Barbour A & Birkbeck T.H.

(1995) Stimulation or inhibition of growth of the

unicel-lular alga Pavlova lutheri by bacteria isolated from larval

turbot culture systems Journal of Applied Bacteriology

79, 519^524.

Munro P.D., Henderson R.J., Barbour A & Birbeck T.H.

(1999) Partial decontamination of rotifers with ultraviolet

radiation: the e¡ect of changes in the bacterial load and

£ora of rotifers on mortalities in start-feeding larval

tur-bot Aquaculture 170, 229^244.

Naas K., Huse I & Iglesias J (1996) Illumination in ¢rst

feed-ing tanks for marine ¢sh larvae Aquacultural Engineerfeed-ing

15, 291^300.

Naas K.E., Naess T & Harboe T (1992) Enhanced 1st feeding

of halibut Larvae (Hippoglossus hippoglossus L) in green

water Aquaculture 105, 143^156.

Ns T & Bergh O (1994) Calanoid copepod resting eggs can

be surface-disinfected Aquacultural Engineering 13, 1^9.

Nss T., Germain Henry M & Naas K.E (1995) First feeding

of Atlantic halibut (Hippoglossus hippoglossus) using

dif-ferent combinations of Artemia and wild zooplankton.

Aquaculture 130, 235^250.

Nagata W.D & White J.N.C (1992) E¡ects of yeast and algal

diets on growth and biochemical composition of the

roti-fer Brachionus plicatilis (Mˇller) in culture Aquaculture

and Fisheries Management 23, 13^21.

Navarro J.C., Amat F & Sargent J.R (1991) A study of the

var-iations in lipid levels, lipid class composition and fatty acid

composition in the ¢rst stages of Artemia sp Marine

Biol-ogy 111, 461^465.

Navarro J.C., Amat F & Sargent J.R (1993) The lipids of the

cysts of fresh-water and marine-type Artemia

Aquacul-ture 109, 327^336.

Navarro J.C., Henderson R.J., McEvoy L.A., Bell M.V & Amat

F (1999) Lipid conversions during enrichment of Artemia.

Aquaculture 174, 155^166.

Navarro N & Sarasquete C (1998) Use of freeze-dried

micro-algae for rearing gilthead seabream, Sparus aurata,

lar-vaeçI Growth, histology and water quality Aquaculture

167, 179^193.

Navarro N & Yu¤fera M (1998) In£uence of the food ration

and individual density on production e⁄ciency of

semi-continuous cultures of Brachionus fed microalgae dry

powder Hydrobiologia 387/388, 483^487.

Nicolas J.L., Robic E & Ansquer D (1989) Bacterial £ora

as-sociated with a trophic chain consisting of microalgae,

ro-tifers and turbot larvaeçin£uence of bacteria on larval

survival Aquaculture 83, 237^248.

Nordgreen A., Hamre K & Langdon C (2007) Development

of lipid microbeads for delivery of lipid and water-soluble

materials to Artemia Aquaculture 273, 614^623.

Ogle J.T., Lemus J.T., Nicholson L.C., Barnes D.N & Lotz J.M (2005) Characterization of an extensive zooplankton cul- ture system coupled with intensive larval rearing of red snapper Lutjanus campechanus in Copepods in Aquaculture (ed by C.-S Lee, P.J O’Bryen & N.H Marcus), pp 225^244 Blackwell Publishing, Oxford, UK.

ie G., Reitan K.I & Olsen Y (1994) Comparison of rotifer culture quality with yeast plus oil and algal-based culti- vation diets Aquaculture International 2, 225^238.

ie G., Makridis P., Reitan K.I & Olsen Y (1997) Protein and carbon utilization of rotifers (Brachionus plicatilis) in ¢rst feeding of turbot larvae (Scophthalmus maximus L.) Aqua- culture 153, 103^122.

Olsen A.I., OlsenY., Attramadal Y., Christie K., Birkbeck T.H., Skjermo J & Vadstein O (2000) E¡ect of short term feeding of microalgae on the bacterial £ora associated with juvenile Artemia franciscana Aquaculture 190, 11^25.

Olsen Y (2004) Live food technology of cold-water marine

¢sh larvae in Culture of Cold-Water Marine Fish (ed by

E Mokness, E Kjrsvik & Y Olsen), pp 73^128 Blackwell Publishing, Oxford, UK.

Papakostas S., Dooms S.,Triantafyllidis A., Deloof D., Kappas I., Dierckens K., De Wolf T., Bossier P., Vadstein O., Kui S., Sorgeloos P & Abatzopoulos T.J (2006) Evaluation of DNA methodologies in identifying Brachionus species used in European hatcheries Aquaculture 255, 557^564 Park H.G., Lee K.W., Cho S.H., Kim H.S., Jung M.M & Kim H.S (2001) Possibility of high density culture of fresh- water rotifer, Brachionus calyci£orus Hydrobiologia 446/

Payne M.F & Rippingale R.J (2001) E¡ects of salinity, cold storage and enrichment on the calanoid copepod Gladio- ferens imparipes Aquaculture 201, 251^262.

Polo A.,Yu¤fera M & Pascual E (1992) Feeding and growth of gilthead seabream (Sparus aurata L) larvae in relation to the size of the rotifer strain used as food Aquaculture

103, 45^54.

Rainuzzo J.R., Reitan K.I & Olsen Y (1997) The signi¢cance

of lipids at early stages of marine ¢sh: a review ture 155, 105^118.

Aquacul-Rajkumar M & Vasagam K (2006) Suitability of the pod, Acartia clausi as a live feed for Seabass larvae (Lates calcarifer Bloch): compared to traditional live-food organ- isms with special emphasis on the nutritional value Aquaculture 261, 649^658.

cope-Rawlings T.K., Ruiz G.M & Colwell R.R (2007) Association

of Vibrio cholerae O1El Tor and O139 Bengal with the

cope-pods Acartia tonsa and Eurytemora a⁄nis Applied and

En-vironmental Microbiology 73,7926^7933.

Reitan K.I., Rainuzzo J.R., ie G & Olsen Y (1993)

Nutri-tional e¡ects of algal addition in ¢rst-feeding of turbot

(Scophthalmus maximus L.) larvae Aquaculture 118, 257^

275.

Reitan K.I., Rainuzzo J.R & Olsen Y (1994) In£uence of

lipid composition of live feed on growth, survival and

pigmentation of turbot larvae Aquaculture International

2, 33^48.

Reitan K.I., Rainuzzo J.R., ie G & OlsenY (1997) A review of

the nutritional e¡ects of algae in marine ¢sh larvae

Aqua-culture 155, 207^221.

Rhodes A & Boyd L (2005) Formulated feeds for

harpacti-coid copepods: implications for population growth and

fatty acid composition In: Copepods in Aquaculture (ed.

by C.-S Lee, P.J O’Bryen & N.H Marcus), pp 61^73

Black-well Publishing, Oxford, UK.

Ritar A.J., Dunstan G.A., Nelson M.M., Brown M.R., Nichols

P.D., Thomas G.W., Smith E.G., Crear B.J & Kolkovski S.

(2004) Nutritional and bacterial pro¢les of juvenile

Arte-mia fed di¡erent enrichments and during starvation.

Aquaculture 239, 351^373.

Rocha R., Ribeiro L., Costa R & Dinis M.T (2008) Does the

presence of microalgae in£uence ¢sh larvae prey capture?

Aquaculture Research 39, 362^369.

Rodr|¤guez C., Pe¤rez J.A., Bad|¤a P., Izquierdo M.S.,

HernaŁn-dez-Palacios H & Lorenzo A (1998) The n-3 highly

unsa-turated fatty acids requirements of gilthead seabream

(Sparus aurata L.) larvae when using an appropriate

DHA/EPA ratio in the diet Aquaculture 196, 9^23.

Rombaut G., Dhert P., Vandenerghe J., Verschuere L.,

Sorge-loos P & Verstraete W (1999) Selection of bacteria

enhan-cing the growth rate of exenically hatched rotifers

(Brachionus plicatilis) Aquaculture 176, 195^207.

Rombaut G., Suantika G., Boon N., Maertens S., Dhert P.,Top

E., Sorgeloos P & Verstraete W (2001) Monitoring of the

evolving diversity of the microbial community present in

rotifer cultures Aquaculture 198, 237^252.

Rnnestad I & Conceicao L.E.C (2005) Aspects of protein

and amino acids digestion and utilization by marine ¢sh

larvae in Physiological and Ecological Adaptations to

Feed-ing in Vertebrates (ed by J.M Starck & T Wang), pp 389^

416 Science Publishers, En¢eld, NH, USA.

Rnnestad I.,Tonheim S.K., Fyhn H.J., Rojas-Garcia C.R.,

Ka-misaka Y., Koven W., Finn R.N., Terjesen B.F., Barr Y &

Conceicao L.E.C (2003) The supply of amino acids during

early feeding stages of marine ¢sh larvae: a review of

re-cent ¢ndings Aquaculture 227, 147^164.

Rosenlund G & Halldo¤rsson OŁ (2007) Cod juvenile

produc-tion: research and commercial developments Aquaculture

268, 188^194.

Rosenlund G., Stoss J & Talbot C (1997) Co-feeding marine

¢sh larvae with inert and live diets Aquaculture 155,

183^191.

Saavedra M., Conceicao L.E.C., Pousao-Ferreira P & Dinis M.T (2006) Amino acid pro¢les of Diplodus sargus (L., 1758) larvae: implications for feed formulation Aquacul- ture 261, 587^593.

Saavedra M., Beltran M., Pousao-Ferreira P., Dinis M.T.,

Blas-co J & Conceicao L.E.C (2007) Evaluation of ity of individual amino acids in Diplodus puntazzo larvae: towards the ideal dietary amino acid pro¢le Aquaculture

bioavailabil-263, 192^198.

Saavedra M., Pousao-Ferreira P.,Yu¤fera M., Dinis M.T & ceicao L.E.C (2009) A balanced amino acid diet improves Diplodus sargus larval quality and reduces nitrogen excre- tion Aquaculture Nutrition in press.

Con-Sainz-Hernandez J.C & Maeda-Martinez A.N (2005) Sources of Vibrio bacteria in mollusc hatcheries and control methods: a case study Aquaculture Research 36, 1611^1618.

Salvesen I., Reitan K.I., Skjermo J & ie G (2000) Microbial environments in marine larviculture: impacts of algal growth rates on the bacterial load in six microalgae Aquaculture International 8, 275^287.

Sargent J., Bell G., McEvoy L., Tocher D & Estevez A (1999) Recent developments in the essential fatty acid nutrition

of ¢sh Aquaculture 177, 191^199.

Sargent J., McEvoy L., Estevez A., Bell G., Bell M., Henderson

J & Tocher D (1999) Lipid nutrition of marine ¢sh during early development: current status and future directions Aquaculture 179, 217^229.

Schlechtriem C., Focken U & Becker K (2005) Digestion and assimilation of the free-living nematode Panagrellus redi- vivus fed to ¢rst feeding coregonid larvae: evidence from histological and isotopic studies Journal of theWorld Aqua- culture Society 36, 24^31.

Schluter M., Soeder C.J & Growneweg J (1987) Growth and food conversion of Brachionus rubens in continuos cul- ture Journal of Plankton Research 9,761^783.

Segers H (1997) Nomenclatural consequences of some cent studies on Brachionus plicatilis (Rotifera, Brachioni- dae) Hydrobiologia 313/314, 121^122.

re-Shields R.J., Bell J.G., Luizi F.S., Gara B., Bromage N.R.

& Sargent J.R (1999) Natural copepods are superior

to enriched Artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids Journal of Nutrition 129, 1186^1194.

Sijtsma l & de Swaarf M.E (2004) Biotechnological duction and apllication of w3 polyunsaturated fatty acid docosohexaenoic acid Applied Microbiology and Biotech- nology 64, 146^153.

pro-Skjermo J & Vadstein O (1993) Characterization of the terial £ora of mass cultivated Brachionus plicatilis Hydro- biologia 255/256, 185^191.

bac-Skjermo J & Vadstein O (1999) Techniques for microbial control in the intensive rearing of marine larvae Aquacul- ture 177, 333^343.

Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

Snell T.W & Carrillo K (1984) Body size variation among

strains of the rotifer Brachionus plicatilis Aquaculture 37,

359^367.

Sochard M.R., Wilson D.F., Austin B & Colwell R.R (1979)

Bacteria associated with the surface and gut of marine

copepods Applied and Environmental Microbiology 37,

750^759.

Song X., Zhang X., Guo N., Zhu L & Kuang C (2007)

Assess-ment of marine thraustochytrid Schizochytrium

limaci-num OUC88 for mariculture by enriched feeds Fisheries

Science 73, 565^573.

Sorgeloos P., Dhert P & Candreva P (2001) Use of the brine

shrimp, Artemia spp., in marine ¢sh larviculture

Aqua-culture 200, 147^159.

Sorokin J & Panov D (1966) The use of C14 for the

quantita-tive study of the nutrition of ¢sh larvae International

Re-view of Hydrobiology 51,743^756.

Srivastava A., Hamre K & Stoss J (2006) Protein content

and amino acid composition of the live feed rotifer

(Bra-chionus plicatilis): with emphasis on the water soluble

fraction Aquaculture 254, 534^543.

Stttrup J.G (2003) Production and nutrition value of

cope-pods in Live Feeds in MarineAquaculture (ed by J.G

Stttr-up & L.A McEvoy), pp 145^205 Blackwell Publishing,

Oxford, UK.

Stttrup J.G., Shields R., Gillespie M., Gara M.B., Sargent J.R.,

Bell J.G., Henderson R.J.,Tocher D.R., Sutherland R., Nss

T., Mangor-Jensen A., Naas K., van der Meeren T., Harboe

T., Sanchez F.J., Sorgeloos P., Dhert P & Fitzgerald

R (1998) The production and use of copepods in larval

rearing of halibut, turbot and cod Bulletin of the

Aquacul-ture Association of Canada 4, 41^45.

Su H.-M., Cheng S.-H., Chen T.-I & Su M.-S (2005) Culture of

copepods and applications to marine ¢n¢sh larval rearing

in Taiwan in Copepods in Aquaculture (ed by C.-S Lee, P.J.

O’Bryen & N.H Marcus), pp 11^24 Blackwell Publishing,

Oxford, UK.

Suantika G., Dhert P., Nurhudah M & Sorgeloos P (2000)

High-density production of the rotifer Brachionus

plicati-lis in a recirculation system: consideration of water

qual-ity, zootechnical and nutritional aspects Aquaculture

Engineering 21, 201^214.

Suantika G., Dhert P., Rombaut G.,Vandenberghe J., De Wolf

T & Sorgeloos P (2001) The use of ozone in a high density

recirculation system for rotifers Aquaculture 201, 35^49.

Suatoni E., Vicario S., Rice S., Snell T & Caccone A (2006)

An analysis of species boundaries and biogeographic

pat-terns in a cryptic complex: the rotifer Brachionus plicatilis.

Molecular Phylogenetics and Evolution 41, 86^98.

Suminto H.K & Hirayama K (1997) Application of a

growth-promoting bacteria for stable mass culture of three

mar-ine microalgae Hydrobiologia 358, 223^230.

Szyper J.P (1989) Nutritional depletion of the aquaculture

feed organisms Euterpina acutifrons, Artemia sp and

Bra-chionus plicatilis during starvation Journal of the World

Aquaculture Society 20, 162^169.

Tanasomwang V & Muroga K (1992) E¡ect of sodium furstyrenate on the reduction of bacterial contamina- tion of rotifers (Brachionus plicatilis) Aquaculture 103, 221^228.

ni-Tendencia E.A & dela Pena M (2003) Investigation of some components of the greenwater system which makes it ef- fective in the control of luminous bacteria Aquaculture

218, 115^119.

Theilacker G.H & McMaster M.F (1971) Mass culture of the rotifer Brachionus plicatilis and its evaluation as food for larval anchovies Marine Biology 10, 183^188.

Toledo J.D., Golez M.S., Doi M & Ohno A (1999) Use of pod nauplii during early feeding stage of grouper Epine- phelus coioides Fisheries Science 65, 390^397.

cope-Toledo J.D., Golez M.S & Ohno A (2005) Studies on the use of copepods in the semi-intensive seed production of group-

er Epinephelus coiodes in Copepods in Aquaculture (ed by C.-S Lee, P.J O’Bryen & N.H Marcus), pp.11^24 Blackwell Publishing, Oxford, UK.

Tonheim S.K., KovenW & Roennestad I (2000) Enrichment

of Artemia with free methionine Aquaculture 190, 223^235.

Tovar D., Zambonino J., Cahu C., Gatesoupe F.J., JuaŁrez R & Le¤sel R (2002) E¡ect of live yeast incorpora- tion in compound diet on digestive enzyme activity in sea bass (Dicentrarchus labrax) larvae Aquaculture 204, 113^123.

VaŁzquez-Turk P.E., Krejci M.E & Yang W.T (1982) A laboratory

meth-od for the culture of Acartia tonsa (Crustacea: Copepmeth-oda) using rice bran Journal of Aquariculture and Aquatic Sciences 3, 25^27.

Vadstein O., ie G & Olsen Y (1993) Particle size dependent feeding by the rotifer Brachionus plicatilis Hydrobiologia 255/256, 261^267.

van der Meeren T & Naas K.E (1997) Development of rearing techniques using large enclosed ecosystems in the mass production of marine ¢sh fry Reviews in Fishesries Science

5, 367^390.

van der Meeren T., Olsen R.E., Hamre K & Fyhn H.J (2008) Biochemical composition of copepods for evaluation of feed quality in production of juvenile marine ¢sh Aqua- culture 274, 375^397.

Van Stappen G (1996a) Introduction, biology and ecology of Artemia in Manual on the Production and Use of Live Food for Aquaculture FAO Fisheries Technical Paper (ed by

P Lavens & P Sorgeloos), pp.79^106 FAO, Rome, Italy Van Stappen G (1996b) Use of cysts in Manual on the Produc- tion and Use of Live Food forAquaculture FisheriesTechnical Paper (ed by P Lavens & P Sorgeloos), pp 107^136 FAO, Rome, Italy.

Vega-Orellana O.M., Fracalossi D.M & Sugai J.K (2006) Dourado (Salminus brasiliensis) larviculture: weaning and ontogenetic development of digestive proteinases Aquaculture 252, 484^493.

Verner-Je¡reys D.W., Shields R.J & Birkbeck T.H (2003) Bacterial in£uences of Atlantic halibut Hippoglossus

hippoglossus yolk-sac larval survival and start-feed

re-sponse Diseases of Aquatic Organisms 56, 105^113.

Verschuere L., Rombaut G., Huys G., Dhont J., Sorgeloos P &

Verstraete W (1999) Microbial control of the culture of

Artemia juveniles through preemptive colonization by

selected bacterial strains Applied and Environmental

Mi-crobiology 65, 2527^2533.

Villalta M., Este¤vez A & Bransden M (2005) Arachidonic

acid enriched live prey induces albinism in Senegal sole

(Solea senegalensis) larvae Aquaculture 245, 193^209.

Villalta M., Este¤vez A., Bransden M.P & Bell J.G (2005) The

e¡ect of graded concentrations of dietary DHA on growth,

survival and tissue fatty acid pro¢le of Senegal sole (Solea

senegalensis) larvae during the Artemia feeding period.

Aquaculture 249, 353^365.

Villalta M., Este¤vez A., Bransden M.P & Bell J.G (2008a)

Ara-chidonic acid, araAra-chidonic/eicosapentaenoic acid ratio,

stearidonic acid and eicosanoids are involved in

dietary-induced albinism in Senegal sole (Solea senegalensis).

Aquaculture Nutrition 14, 120^128.

Villalta M., Este¤vez A., Bransden M.P & Bell J.G (2008b)

Ef-fects of dietary eicosapentaenoic acid on growth,

survi-val, pigmentation and fatty acid composition in Senegal

sole (Solea senegalensis) larvae during the Artemia feeding

period Aquaculture Nutrition 14, 232^241.

Villamil L., Figueras A., Planas M & Novoa B (2003) Control

of Vibrio alginolyticus in Artemia culture by treatment

with bacterial probiotics Aquaculture 219, 43^56.

Villamil L., Figueras A., Toranzo A.E., Planas M & Novoa B.

(2003) Isolation of a highly pathogenic Vibrio pelagius

strain associated with mass mortalities of turbot,

Scophthalmus maximus (L.), larvae Journal of Fish Diseases

26, 293^303.

Walford J & Lam T.J (1987) E¡ects of feeding with

microcap-sules on the content of essential fatty acids in live foods for

the larvae of marine ¢shes Aquaculture 61, 219^229.

Walz N (1993) Characteristics of two-stage chemostat

cul-tures of Brachionus angularis in Plankton Regulation

Dy-namics (ed by N.Walz), pp 62^76 Springer-Verlag, Berlin,

Germany.

Watanabe T., Kitajima C & Fujita S (1983) Nutritional values

of live organisms used in Japan for mass propagation of

¢shça review Aquaculture 34, 115^143.

Yu¤fera M (1982) Morphometric characterisation of a

small-sized strain of Brachionus plicatilis in culture Aquaculture

27, 55^61.

Yu¤fera M (1987) E¡ects of algal diet and and temperature on

the embryonic development time of the rotifer Brachionus

plicatilis in culture Hydrobiologia 147, 319^322.

Yu¤fera M (2001) Studies on Brachionus (Rotifera): an ple of interaction between fundamental and applied re- search Hydrobiologia 446/447, 383^392.

exam-Yu¤fera M & LubiaŁn L.M (1990) E¡ects of microalgal diet on growth and development of invertebrates in marine aqua- culture in An Introduction to Applied Phycology (ed by I Akatsuka), pp 209^227 An Introduction to Applied Phy- cology,The Hague, the Netherlands.

Yu¤fera M & Navarro N (1995) Population growth dynamics

of the rotifer Brachionus plicatilis cultured in non-limiting food condition Hydrobiologia 313/314, 399^405 Yu¤fera M & Pascual E (1980) Estudio del rendimiento de cultivos masivos del rot|¤fero Brachionus plicatilis alimen- tados con levadura de pani¢cacio¤n Investigacion Pesquera

44, 55^61.

Yu¤fera M & Pascual E (1989) Biomass and elemental position (C.N.H.) of the rotifer Brachionus plicatilis cul- tured as larval food Hydrobiologia 186/187, 371^374 Yu¤fera M., LubiaŁn L.M & Pascual E (1983) Efecto de cuatro algas marinas sobre el crecimiento poblacional de dos ce- pas de Brachionus plicatilis (Rotifera: Brachionidae) en cultivo Investigacion Pesquera 47, 325^337.

com-Yu¤fera M., Rodr|¤guez A & LubiaŁn L.M (1984) Zooplankton ingestion and feeding behavior of Penaeus kerathurus lar- vae reared in the laboratory Aquaculture 42, 217^224 Yu¤fera M., Parra G & Pascual E (1997) Energy content of the rotifers Brachionus plicatilis and Brachionus rotundiformis

in relation to temperature Hydrobiologia 358, 83^87 Yu¤fera M., Kolkovski S., FernaŁndez-D|¤az C., Rinchard J., Lee K.J & Dabrowski K (2003) Delivering bioactive com- pounds to ¢sh larvae using microencapsulated diets Aquaculture 227, 277^291.

Yamasaki T., Aki T., Mori Y., Yamamoto T., Shinozaki M., Kawamoto S & Onu K (2007) Nutritional enrichment

of larval ¢sh feed with Thraustochytrid producing polyunsaturated fatty acids and xanthophylls Journal of Bioscience and Bioengineering 104, 200^206.

Yoshimura K., Hagiwara A., Yoshimatsu T & Kitajima T (1996) Culture technology marine rotifers and the impli- cation for intensive culture of marine ¢sh in Japan Mar- ine and Freshwater Research 47, 217^222.

Yoshimura K.,Tanaka K & Yoshimatsu T (2001) A novel tem for the ultra-high-density production of the rotifer, Brachionus rotundiformisça preliminary report Aquacul- ture 227, 165^172.

sys-Zmora O & Richmond A (2004) Microalgae for aquaculture Microalgae production for aquaculture in Microalgal Cul- ture Biotechnology and Applied Phycology (ed by A Rich- mond), pp 365^379 Blackwell Science, Pondicherry, India Live feeds for ¢sh larvae L E C Conceicao et al Aquaculture Research, 2010, 41, 613^640

r 2009 The Authors

REVIEW ARTICLE

Transport of di- and tripeptides in teleost fish intestine

Tiziano Verri1, Alessandro Romano1, Amilcare Barca1, Gabor Kottra2, Hannelore Daniel2&Carlo Storelli1

1 Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento (formerly University of Lecce), Lecce, Italy

2 Molecular Nutrition Unit, Nutrition and Food Research Center, Technical University of Munich, Freising-Weihenstephan, Germany

Correspondence: T Verri, Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento (formerly University of Lecce),Via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy E-mail: tiziano.verri@unile.it

Abstract

The initial observation of peptide absorption in ¢sh

intestine dates back to 1981, when, in rainbow trout

(Oncorhynchus mykiss), the rate of intestinal

absorp-tion of the dipeptide glycylglycine (Gly-Gly) was

com-pared in vivo with the rate of absorption of its

component amino acid glycine (Gly) The description

of the identi¢cation of the underlying mechanisms

that allow di- and tripeptide transport across the

plasma membranes in ¢sh was provided in 1991,

when the ¢rst evidence of peptide transport activity

was reported in brush-border membrane vesicles of

intestinal epithelial cells of Mozambique tilapia

(Oreochromis mossambicus) by monitoring uptake

of radiolabelled glycyl-L-phenylalanine (Gly-L-Phe)

Since then, the existence of a carrier-mediated, H1

-dependent transport of di- and tripeptides (H1

/pep-tide cotransport) in the brush-border membrane of

¢sh enterocytes has been con¢rmed in many teleost

species by a variety of biochemical approaches,

pro-viding basic kinetics and substrate speci¢cities of

the transport activity In 2003, the ¢rst peptide

trans-porter from a teleost ¢sh, i.e the zebra¢sh (Danio

re-rio) PEPtide transporter 1 (PEPT1), was cloned and

functionally characterized in the Xenopus laevis

oo-cyte expression system as a

low-a⁄nity/high-capa-city system PEPT1 is the protein in brush-border

membranes responsible for translocation of intact

di- and tripeptides released from dietary protein by

luminal and membrane-bound proteases and

pepti-dases The transporter possesses a⁄nities for the

peptide substrates in the 0.1^10 mM range,

depend-ing on the structure and physicochemical nature ofthe substrates After the molecular and functionalcharacterization of the zebra¢sh transporter, the in-terest in PEPT1 in teleost ¢sh has increased and ap-proaches for cloning and functional characteriza-tion of PEPT1 orthologues from other ¢sh species,some of them of the highest commercial value, arenow underway In this paper, we provide a brief over-view of the transport of di- and tripeptides in teleost

¢sh intestine by recalling the bulk of biochemical,biophysical and physiological observations collected

in the pre-cloning era and by recapitulating the morerecent molecular and functional data

Keywords: gut, protein digestion, peptide tion, peptide transport, di- and tripeptides, PEPtidetransporter 1 (PEPT1)/SoLute Carrier 15 familymember A1 (SLC15A1)

absorp-IntroductionHydrolysis of dietary proteins leads to high levels ofshort-chain peptides (di- and tripeptides) in the in-testinal lumen during the digestive processes Thedi- and tripeptides released are either further hydro-lysed to their constituent amino acids or directly ta-ken up in intact form into intestinal epithelial cells.Following apical in£ux, di- and tripeptides are se-quentially hydrolysed by multiple cytosolic hydro-lases (di- and tripeptidases), followed by basolaterale¥ux of the resulting amino acids via di¡erent aminoacid-transporting systems Peptides not undergoing

hydrolysis can exit the cell by a basolateral

peptide-transporting system, not yet identi¢ed on a

molecu-lar basis, and/or by other basolateral solute transport

systems that have occasionally been shown to allow

transport of selected peptides (for a comprehensive

review, see e.g Daniel 2004)

At the apical membrane of enterocytes, transport

of di- and tripeptides is mediated by a single carrier

system, called PEPT1 (PEPtide transporter 1) or

SLC15A1 (SoLute Carrier 15 family member A1), after

classi¢cation of membrane transporters by the

Hu-man Genome Organization Nomenclature

Commit-tee (for a recent review of the transporters of the

SLC15 family, see e.g Daniel & Kottra 2004) PEPT1

functions as an Na1-independent, H1-dependent

transporter for a large variety of di- and tripeptides

Neither free amino acids nor peptides containing

four or more amino acids are accepted as substrates

The transport of di- and tripeptides is electrogenic

and responds to both an inwardly directed

trans-membrane H1gradient (pHoutopHin) and (internal

negative) a transmembrane electrical potential (for

details, see Daniel 2004) Transport is

enantio-selec-tive and involves a variable proton-to-substrate

stoi-chiometry for uptake of neutral and mono- or

polyvalently charged peptides PEPT1 is also

respon-sible for the transport of orally active drugs, such as

b-lactam antibiotics, aminopeptidase and

angioten-sin-converting enzyme (ACE) inhibitors,

d-aminole-vulinic acid and many selected pro-drugs (for a

review, see e.g Rubio-Aliaga & Daniel 2002),

although the option that ACE inhibitors may simply

interact without being transported has recently been

put forward (Knˇtter,Wollesky, Kottra, Hahn, Fischer,

Zebisch, Neubert, Daniel & Brandsch 2008)

Transport of di- and tripeptides in teleost

fish intestine: the pre-cloning era

Transport at the luminal barrier

(brush-border membrane) of ¢sh enterocytes

Peptide transport in ¢sh has been described in detail

in brush-border membranes of European eel (Anguilla

anguilla) intestine (Verri, Ma⁄a & Storelli 1992; Ma⁄a,

Verri, Danieli, Thamotharan, Pastore, Ahearn &

Stor-elli 1997; Verri, Ma⁄a, Danieli, Herget, Wenzel, Daniel

& Storelli 2000; Verri, Danieli, Bakke, Romano, Barca,

Rnnestad, Ma⁄a & Storelli 2008), Mozambique

tila-pia (Oreochromis mossambicus) intestine (Reshkin &

Ahearn 1991; Thamotharan, Gomme, Zonno, Ma⁄a,

Storelli & Ahearn1996), copper rock¢sh (Sebastes

caur-inus) intestine and pyloric ceca (Thamotharan &Gomme et al 1996), Atlantic salmon (Salmo salar) in-testine and pyloric ceca (Bakke-McKellep, Nordrum,Krogdahl & Buddington 2000; Nordrum, Bakke-McKellep, Krogdahl & Buddington 2000), rainbowtrout (Oncorhynchus mykiss) intestine and pyloric ceca(Boge¤, Rigal & Peres 1981; Buddington & Diamond

1986, 1987; Nordrum et al 2000) and Antarctic dile ice¢sh (Chionodraco hamatus) intestine (Ma⁄a,Rizzello, Acierno, Verri, Rollo, Danieli, D˛ring, Daniel

croco-& Storelli 2003) Interestingly, some of these early dies pointed out that in ¢sh, as in mammals (for a re-cent review, see e.g Daniel 2004), amino acids inpeptide-bound form can be absorbed more e⁄cientlythan the mixture of the constituent amino acids (Boge¤

stu-et al.1981; Reshkin & Ahearn 1991)

In ¢sh brush-border membrane vesicle (BBMV) parations [a BBMV preparation is a highly puri¢edfraction of brush-border membranes mostly consist-ing (up to 80%) of closed and right side out orientedvesicles Brush-border membrane vesicles are suitablefor a variety of in vitro studies, among which trans-membrane transport experiments (for details, see e.g.Biber, Stieger, Stange & Murer 2007, and literature ci-ted therein)], carrier-mediated uptake of radiolabelledpeptides is a saturable process with Michaelis^Mentenkinetics, and is stimulated by a transmembrane elec-trical potential and to a lesser extent by an inwardlydirected transmembrane H1 gradient (Reshkin &Ahearn 1991;Thamotharan & Gomme et al.1996; Maf-

pre-¢a et al 1997; Verri et al 2000) Interestingly, in BBMV,intravesicular acidi¢cation is observed with the addi-tion of di- and tripeptides to the extravesicular med-ium (Verri et al 1992, 2000; Ma⁄a et al 1997, 2003).Furthermore, due to the electrogenic nature of thetransport ^ e.g the transport of the peptide is coupled

to the transfer of positive charge(s) from the external

to the internal side of the membrane ^ addition of and tripeptides to the extravesicular medium inducessigni¢cant membrane potential depolarization (Verri

di-et al 2008) Didi-ethylpyrocarbonate (DEP), which ciently inhibits peptide transport in mammalianBBMV (Miyamoto, Ganapathy & Leibach 1986; Kra-mer, Girbig, Petzoldt & Leipe 1988; Kato, Maegawa,Okano, Inui & Hori 1989), also inhibits peptide trans-port in ¢sh BBMV (Verri et al 1992, 2000, 2008; Tha-motharan & Gomme et al 1996; Ma⁄a et al 1997,2003) Taken together, the BBMV data provide a strongbiochemical basis for the understanding of the car-rier-mediated mechanism that allows di- and tripep-tide uptake across the apical barrier of teleost ¢shenterocytes, and establish that a single carrier systemDi- and tripeptide transport in ¢sh gut T.Verri et al Aquaculture Research, 2010, 41, 641^653

e⁄-r 2009 Blackwell Munksgaard

with apparent Kmvalues (Km,app) (Km,appindicates the

apparent concentration of di- or tripeptide that yields

one-half of maximal in£ux) in the 0.1^10 mM range

mediates the transport process This system transports

a large variety of di- and tripeptides, with remarkable

di¡erences in substrate a⁄nity and maximal velocity

depending on the particular amino acid composition

of the peptide substrate used for assay As in

mamma-lian systems, the transport is stereoselective, with a

preference forL-a amino acids, although peptides

con-tainingD-isomers of amino acids (such asD-Phe-L-Ala)

are also accepted (Verri et al 2000; Ma⁄a et al 2003),

their a⁄nity for interaction largely depending on the

location of the D-amino acid residue in the peptide

backbone and the polarity of the amino acid

side chain (see e.g Daniel 2004) As assessed by

cis-in-hibition experiments, two di¡erent peptides that are

si-multaneously present in the extravesicular space

compete for the same transport system (Reshkin &

Ahearn 1991;Thamotharan & Gomme et al.1996;

Maf-¢a et al 1997; Verri et al 2000) The same holds true for

the presence of a peptide and a peptide-like drug (such

as the aminocephalosporin antibiotic cephalexin;

Ma⁄a et al 1997) As a general observation, in kinetic

experiments in which BBMVs are used in conjunction

with a radioactive peptide, a linear apparent di¡usion

process having a rate that is proportional to the

extra-vesicular peptide concentration is always evident

besides the saturable (Michaelis^Menten type)

carrier-mediated component (Reshkin & Ahearn

1991; Thamotharan & Gomme et al 1996; Ma⁄a et al

1997; Verri et al 2000) This di¡usive pathway, which

may include a carrier-independent in£ux and/or very

low-a⁄nity, high-capacity carrier systems (as gested by Thamotharan & Gomme et al 1996), ac-counts for a signi¢cant fraction of the peptide uptake

sug-in teleost ¢sh BBMV

The rates of dipeptide absorption by ¢sh intestinehave also been measured using intact tissue of Atlan-tic salmon and rainbow trout (Bakke-McKellep et al.2000; Nordrum et al 2000) in conjunction with theeverted sleeve method (Karasov & Diamond1983; Bud-dington, Chen & Diamond 1987) (By mounting the in-testinal sleeve on a grooved rod, this method allowsisolation of the serosal surface from the incubationmedium, so that one measures only uptake across theapical membrane rather than across both cell faces si-multaneously In addition, sleeves from di¡erent seg-ments of the alimentary canal can be mounted ondi¡erent rods, thus allowing regional analysis of nutri-ent uptake.) In these experimental setups, in bothAtlantic salmon and rainbow trout peptide transportacross the brush-border membrane barrier appears

as a combination of carrier-mediated and apparent fusion processes, the latter probably accounting forthe majority of total uptake at higher concentrations

dif-of peptides (Bakke-McKellep et al 2000; Nordrum

et al 2000) In salmonids, there is a declining mal-to-distal gradient of peptide absorption along thepost-gastric intestinal tract (pyloric ceca4proximalintestine4mid-intestine4distal intestine), which issimilar to that found for absorption of both glucoseand amino acids (Buddington & Diamond 1986, 1987;Bakke-McKellep et al 2000; Nordrum et al 2000).Furthermore, in each region, the rates of peptide up-take fall within the same order of magnitude as those

Carnosine0.0

0.51.01.52.02.5

Proximal intestineMid intestineDistal intestine

of amino acid uptake (Fig 1) The basic features of the

carrier-mediated peptide transport process occurring

at the brush-border membrane of teleost ¢sh

absorb-ing intestinal cells are summarized in Table 1 and in

the literature cited therein

Transport at the contraluminal barrier

(basolateral membrane) of ¢sh enterocytes

To our knowledge, only one paper has focused on the

pathway for peptide transport across the basolateral

membrane of teleost ¢sh enterocytes Using

basolat-eral membrane (BLMV) preparations in conjunction

with the radioactive tracer [14C]Gly-Sar,

Thamothar-an, Zonno, Storelli and Ahearn (1996) have detected

Gly-Sar transport at the basolateral membrane of

Mozambique tilapia enterocytes Kinetic analysis of

the basolateral transport rate revealed that the

trans-port occurs by a saturable process conforming to

Mi-chaelis^Menten kinetics, with a Km,appfor Gly-Sar of

13.3 3.8 mM This transport activity is almost

in-sensitive to DEP Furthermore, [14C]Gly-Sar in£ux

into tilapia BLMV shows cis-inhibition by several

other dipeptides

In summary, transcellular transport of peptides

across teleost ¢sh post-gastric intestinal barrier can

be accounted for by a combination of carrier-mediated

and apparent di¡usion routes At the brush-border

membrane level, peptide transport occurs by a

well-characterized carrier-mediated process in

conjunc-tion with an apparently di¡usive process, which may

include (a) carrier-independent in£ux and/or putative

very low-a⁄nity, high-capacity carrier system(s)

Ab-sorbed di- and tripeptides may be hydrolysed to

com-ponent amino acids within the cell before basolateral

exit via amino acid transport systems or leave the cell

intact via a not yet well characterized basolateral

transporter In intact tissue, paracellular transport of

di- and tripeptides cannot be excluded

Transport of di- and tripeptides in teleost

fish intestine: the cloning era

The zebra¢sh (Danio rerio) PEPT1 transporter

paradigm

In the last decades, the cyprinid D rerio (zebra¢sh), a

small (3^4 cm long) freshwater teleost ¢sh, has

emerged as a powerful model organism for

experi-mental biology, vertebrate embryology,

developmen-tal genetics and toxicology studies In addition, its

importance as an animal model for regulatory andintegrative physiology studies has steadily increased(for a review, see e.g Briggs 2002) In 2003, we suc-ceeded in the molecular cloning of zebra¢sh PEPT1,the ¢rst PEPT1-type peptide transporter from a tele-ost ¢sh (Verri, Kottra, Romano, Tiso, Peric, Ma⁄a,Boll, Argenton, Daniel & Storelli 2003) Zebra¢shPEPT1 complementary DNA (cDNA) (zfPEPT1; Gen-Bank Acc No AY300011) is 2746 bp long, with anopen reading frame of 2157 bp encoding a putativeprotein of 718 amino acids Hydropathy analysis pre-dicts at least 12 potential membrane-spanning do-mains (TMDs) with a large extracellular loopbetween TMDs IX and X The predicted zebra¢shPEPT1 amino acid sequence exhibits a percentage ofidentity of 60.3^61.5% when compared with otherPEPT1-type members of the SLC15 family alreadycharacterized from mammals and birds

As assessed by reverse transcription-polymerasechain reaction (RT-PCR), zebra¢sh PEPT1 mRNA ishighly expressed in the intestine of adult ¢sh, as well

as in the kidney and spleen, although to a lower tent During larval development, PEPT1 mRNA ex-pression starts by 2 days post fertilization (dpf),slightly increases by 3 dpf and reaches the highestobserved values by 4^7 dpf In this time frame, ex-pression is strictly localized at the proximal intestinallevel (i.e the intestinal bulb; for a recent description

ex-of the digestive system development in zebra¢sh, seee.g Ng, de Jong-Curtain, Mawdsley, White, Shin, Ap-pel, Dong, Stainier & Heath 2005), as assessed bywhole-mount in situ hybridization analysis NomRNA signal is detectable in the mid- and posteriorintestine or other tissues/organs of the developingembryo (Verri et al 2003)

To characterize zebra¢sh PEPT1 function, thecomplementary RNA (cRNA) originating fromzfPEPT1has been injected into Xenopus laevis oocytes

to allow biosynthesis and expression of the tional protein at the oocyte plasma membrane [Thisexperimental manoeuvre allows biosynthesis offunctional heterologous proteins by the Xenopus lae-vis oocyte ‘expression system’, which is able to e⁄-ciently transcribe and translate injected geneticinformation, perform assembly of the foreign proteinproducts, correctly process the nascent polypeptidesand target them to the proper subcellular compart-ment of the oocyte (i.e the plasma membrane in thecase of plasma membrane proteins; for a comprehen-sive review, see e.g Romero, Kanai, Gunshin & Hedi-ger 1998).] To establish the basic kinetic properties ofthe zebra¢sh PEPT1 transporter, the zwitterionicDi- and tripeptide transport in ¢sh gut T.Verri et al Aquaculture Research, 2010, 41, 641^653

func-r 2009 Blackwell Munksgaard

Table 1 Characteristics of carrier-mediated peptide transport in teleost ¢sh post-gastric alimentary canal (intestinal) regions

Species

mental

Test substrate

b Gly- L -Ala 0.97  0.42w,z,‰ Verri et al (2000)

b Gly- L -Asn 2.59  0.73w,z,‰ Verri et al (2000)

b Gly-Sar 1.75  0.47w,z,‰ Verri et al (2000)

b L -Pro-Gly 0.87  0.36w,z,‰ Verri et al (2000) Mozambique

tilapiak

(Oreochromis

mossambicus)

Sexually immature adult

Whole intestine a Gly- L -Phe 9.8  3.5w,,‰ Reshkin and Ahearn

(1991)

Upper one-half of the intestine

a Gly-Sar 0.56  0.08w,,‰ Thamotharan,

Gomme et al (1996) Antarctic crocodile

icefish

(Chionodraco

hamatus)

Sexually mature adult

Whole intestine b Gly- L -Pro 0.806  0.161w,ww Maffia et al (2003)

Atlantic salmon

(Salmo salar)

Smolt Pyloric caecazz d Gly- L -Pro 0.5  0.4z,,‰‰ Bakke-McKellep et al.

(2000) Mid-intestinezz d 1.5  0.4z,,‰‰ Bakke-McKellep et al.

(2000)

(2000) Proximal intestinezz d Gly-Sar 8.579  5.327z,z,‰‰ Nordrum et al (2000)

13.120  6.620z,,‰‰ Nordrum et al (2000) 1.370  0.118z,,‰‰,zz Nordrum et al (2000) Rainbow trout

(Oncorhynchus

mykiss)

Smolt Proximal intestinezz d Gly-Sar 9.774  8.736z,z,‰‰ Nordrum et al (2000)

0.747  0.051z,,‰‰ Nordrum et al (2000) 5.270  1.41z,,‰‰,zz Nordrum et al (2000)

K m,app indicates the apparent concentration of dipeptide that yielded one-half of maximal in£ux (J max ) K m,app values are expressed in

mM and are reported as means  SEM of n, number of observations (see references for details) J max data have not been reported in this table as the use of di¡erent experimental approaches make them not comparable (see references for details) Methods: a, brush-border membrane vesicles (BBMV) in conjunction with a radioactive dipeptide to directly monitor dipeptide uptake; b, BBMV in conjunction with the pH-sensitive dye acridine orange to monitor dipeptide-dependent intravesicular acidi¢cation; c, BBMV in conjunction with the voltage-sensitive dye 3,3 0 -diethylthiacarbocyanine iodide [DiS-C 2 (5)] to monitor dipeptide-dependent membrane potential depolariza- tion; d, everted sleeves in conjunction with a radioactive dipeptide to directly monitor dipeptide uptake.

Carnivorous.

wTransport measured in the absence of extravesicular (BBMV) or extracellular (everted sleeves) sodium.

zSeawater-adapted ¢sh.

‰Transport activity measured at 25 1C.

zTransport measured in the presence of extravesicular (BBMV) or extracellular (everted sleeves) sodium.

kOmnivorous (http://www.¢shbase.org).

Freshwater-adapted ¢sh.

wwTransport activity measured at 0 1C.

zzDivision of intestine according to Buddington and Diamond (1987).

‰‰Transport activity measured at 10 1C.

zzSoybean meal-fed ¢sh.

ND, not detected.

dipeptide Gly-L-Gln, a well-known high-a⁄nity

sub-strate for mammalian PEPT1 transporters, has been

used as a test compound in two-electrode voltage

clamp (TEVC) experiments (Verri et al 2003), under

the same experimental conditions as for mammalian

PEPT1 analysis (Kottra & Daniel 2001; Kottra et al

2002) Current^voltage (I^V) relationships (Fig 2)

in-dicate that zebra¢sh PEPT1, similar to the

mamma-lian orthologues, transports electrogenically not

only in the forward but also in the reverse direction,

thus suggesting not only in£ux but also e¥ux of

pep-tides across the plasma membrane, at least under

cer-tain imposed experimental conditions [For instance,

at pH 7.5, the Gly-L-Gln-dependent transport current

is inwardly directed at negative membrane

poten-tials However, the direction of current reverses (i.e

it becomes outwardly directed) at more positive

mem-brane potentials (at  7 and 147 mV when 1 and

20 mM Gly-L-Gln are applied respectively; see Fig 2).]

Gly-L-Gln transport occurs according to Michaelis^

Menten kinetics (in oocytes clamped at  60 mVand

perfused with an uptake solution at pH 7.5, the

kinetic parameters for Gly-L-Gln are K0.551.44

 0.18 mM and Imax5 200 24 nA) [K0.5indicates

the apparent concentration of dipeptide that yields

one-half of maximal transport current (Imax)] and is

strongly a¡ected by both membrane potential (see

Fig 2, where the dependence on membrane potential

is evident, with currents steadily decreasing, passing

from ^160 mV to less negative potentials) and

extra-cellular pH (Table 2) It is noteworthy that the

appar-ent a⁄nity for Gly-L-Gln at pH 5.5 is higher than that

at pH 6.5, the apparent a⁄nity at pH 6.5 is higher

than that at pH 7.5 and these three a⁄nities are

sig-ni¢cantly higher (at least10-fold) than that measured

at pH 8.5 (Table 2) This behaviour is very similar tothat observed with the mammalian transporters(Kottra & Daniel 2001) On the other hand, varyingextracellular pH from 5.5 to 8.5 results, unexpectedly,

in a pronounced increase (at least fourfold) in themaximal transport current (Table 2) This increase

in Imax as the external proton concentration creases is the ¢rst description of such a kinetic beha-viour in any of the PEPT1-type transporters studiedusing the electrophysiological approach Thus, inspite of several similarities, a striking di¡erence ex-ists between the kinetic behaviour of zebra¢sh PEPT1and that of the other known mammalian peptidetransporters

de-Recently, the results obtained with Gly-L-Gln havebeen corroborated using other test peptides for analy-sis Brie£y, zebra¢sh PEPT1 translocates not onlyneutral (Gly-L-Gln) but also acidic (Gly-L-Asp,L-Asp-Gly) and basic (Gly-L-Lys,L-Lys-Gly) dipeptides using

an electrogenic process that follows ten kinetics (similar to that described for Gly-L-Gln;see Table 2) In particular, by comparing pairs of sub-strates with the same charged amino acid residues ineither the amino- or the carboxyl-terminal position

Michaelis^Men-–200 –160 –120 –80 –40 40 80 120

–800–600–400–200

200Membrane potential, V (mV)

1 mM

20 mM

Figure 2 Steady-state current^voltage (I^V)

relation-ships for Gly-L-Gln (1 and 20 mM, pH 7.5) as measured in

zebra¢sh PEPT1-expressing Xenopus laevis oocytes.Values

are means SEM of 10 oocytes

Table 2 Kinetic parameters of di¡erently charged tides (neutral, basic and acidic dipeptides) and their pH dependency

dipep-Substrate pH

K 0.5 (mM,

at ^60 mV)

I max (relative to Gly-Gln at pH 7.5)

No of oocytes (n)

Gly- L -Gln 8.5 9.6  1.6 1.62  0.10 10

7.5 1.44  0.18 1.00 ( 5 200  24 nA) 16 6.5 0.24  0.07 0.43  0.03 10 5.5 0.13  0.03 0.36  0.03 16 Gly- L -Lys 8.5 38  8 1.91  0.21 9

7.5 6.0  1.5 0.94  0.07 17

L -Lys-Gly 8.5 16  6 2.20  0.10 7

7.5 3.5  0.6 1.26  0.07 16 Gly- L -Asp 7.5 21  5 0.64  0.14 8

5.5 0.21  0.03 0.47  0.04 15

L -Asp-Gly 7.5 13  1 0.51  0.07 5

5.5 0.17  0.05 0.36  0.04 5 Electrophysiological data reported in this table were obtained

by experiments conducted on Xenopus laevis oocytes voltage clamped at ^60 mV Peptide-evoked inward currents were mea- sured in the presence of increasing concentrations of dipeptide (0.5^20 mM) in oocytes perfused with solutions at various pH values (ranging from 5.5 to 8.5) K 0.5 indicates the apparent con- centration of dipeptide that yielded one-half of maximal trans- port current (I max ) Values are reported as means  SEM of n, number of oocytes (see last column).

The maximal transport current (I max ) values are relative to I max

of Gly- L -Gln measured at pH 7.5 in the same oocyte.

Di- and tripeptide transport in ¢sh gut T.Verri et al Aquaculture Research, 2010, 41, 641^653

r 2009 Blackwell Munksgaard

of the dipeptide (i.e Gly-L-Asp vs.L-Asp-Gly and Gly-L

-Lys vs.L-Lys-Gly), it has been shown that the position

of the charged amino acid chain in a substrate

signif-icantly a¡ects the transport process Furthermore,

using TEVC experiments combined with intracellular

pH recordings and voltage-clamped tracer

measure-ments (Kottra & Daniel 2001; Kottra et al 2002), the

following evidences have been provided: (a) direct

transport of a peptide across the plasma membrane

of X laevis oocytes, by monitoring the uptake of

radiolabelled Gly-Sar ([14C]Gly-Sar); (b) direct

evi-dence for dipeptide-mediated H1 translocation, by

monitoring zebra¢sh PEPT1-speci¢c intracellular

acidi¢cation; and (c) preliminary £ux coupling

stoichiometry, by comparing the initial rate of

[14C]Gly-Sar uptake with the net charge £ux due to

Gly-Sar-induced inward current In particular,

the unexpected ¢nding of a proton-to-Gly-Sar

coupling coe⁄cient of 0.421 0.035 suggests that

H1/Gly-Sar transport via zebra¢sh PEPT1 may occur

according to a 1:2 stoichiometric ratio (A Romano &

G Kottra, unpubl obs.)

Back to the role of PEPT1 in zebra¢sh

intestinal physiology

The zebra¢sh PEPT1-type peptide transporter

oper-ates in the proximal intestine and medioper-ates the

trans-port of bulk quantities of di- and tripeptides arising

from protein digestion Interestingly, transport of

both neutral and charged peptides exhibits a highly

predictable, large increase with increasing pH, which

is not found in any mammalian PEPT1-type peptide

transporters, and seems to represent a functional

hallmark of the zebra¢sh transporter Also,

expres-sion starts early (2 dpf) during embryo development

This may be placed in the context of the specialties of

zebra¢sh intestinal physiology

Zebra¢sh is a carnivorous ¢sh, which has its

func-tional correlate in the presence of a very short

intest-inal tract Moreover, zebra¢sh is a stomachless ¢sh

(which implies that no acidic content is released into

the proximal intestine) and its intestinal lumen

might be alkaline under normal physiological

condi-tions (due to pancreas and gallbladder secrecondi-tions into

the intestinal lumen) That this holds true is

sup-ported by the ¢nding of alkaline pH (47.5) in adult

zebra¢sh intestinal lumen (Nalbant, Boehmer,

Deh-melt,Wehner & Werner1999) In mammals, the acidic

microclimate layer occurring in the vicinity of the

lu-minal cell surface of the small intestinal epithelium is

important for optimal absorption of peptides viaPEPT1 (see e.g Daniel 2004; Thwaites & Anderson2007) This process depends on the trans-apical pHgradient that is maintained, at least in part, by theactivity of the brush-border membrane Na1/H1ex-changer NHE3 (see e.g Kennedy, Leibach, Ganapa-thy & Thwaites 2002; Thwaites, Kennedy, Raldua,Anderson, Mendoza, Bladen & Simmons 2002) Ithas been shown that NHE3 is not expressed in the in-testine of the Osorezan dace Tribolodon hakonensis(Hirata, Kaneko, Ono, Nakazato, Furukawa, Hasega-

wa, Wakabayashi, Shigekawa, Chang, Romero & ose 2003), a cyprinid taxonomically related tozebra¢sh Therefore, no acidi¢cation process (at leastvia NHE3) might occur in cyprinids and, as a conse-quence, no acidic microclimate pH might be e¡ective,thus making the external cell surface of the intest-inal epithelium fully sense the luminal alkaline pH.[A similar functional arrangement might be e¡ective

Hir-in some other ¢sh species, such as the European eel,

in which the limited stimulatory e¡ect on di- and peptide transport exerted by an inwardly directedtransmembrane H1 gradient (see e.g Ma⁄a et al.1997;Verri et al 2000) strongly correlates with the ab-sence of a brush-border membrane Na1/H1exchan-ger (Vilella, Zonno, Lapadula,Verri & Storelli1995).] Inthis respect, the presence of a peptide transport sys-tem that is adapted to optimally operate at alkaline

tri-pH in the proximal part of the intestine would allowrapid and complete transport of peptides derivedfrom intestinal protein digestion In particular, thepositive modulation by alkaline pH would be highlye¡ective as soon as the alkaline £uids from the pan-creas and gallbladder are released into the proximalintestinal lumen as a consequence of the presence offood in the proximal intestine, thus making the in-crease in the transport activity (see Table 2) func-tional to the rapid removal of the ingested andpartially digested dietary proteins

In the early zebra¢sh embryo, energy requirement

is provided by the yolk until full maturation of thealimentary canal occurs The morphogenesis of thezebra¢sh digestive tract occurs by  2.5 dpf, whencontiguous and histologically recognizable primor-dia of the pharynx, oesophagus and intestine are evi-dent Then, further growth and di¡erentiation of alldigestive organ primordia occur so that by 5 dpf thedigestive system is functional and longitudinallydi¡erentiated in the mouth and oral cavity, pharynx,oesophagus, intestine, rectum and anus (see e.g.Wal-lace & Pack 2003; Ng et al 2005; Wallace, Akhter,Smith, Lorent & Pack 2005) Full opening of the

rostral digestive tract is achieved by  3 dpf, when

the lumen of the posterior pharinx is visible and the

mouth is open The anus is open at 4 dpf Epithelial

polarization occurs parallel to organ morphogenesis,

with markers of apical membranes of the intestinal

cells, e.g.b-actin and alkaline phosphatase, ¢rst

de-tected at  2.5 dpf and increasing from 3 (the end

of the hatching period) to 4 dpf By 4^5 dpf, the

ante-rior intestine also undergoes a transition from a

straight to a coiled tube and exhibits typical folding

(see e.g Wallace & Pack 2003) Zebra¢sh PEPT1 is

al-ready expressed in the proximal intestine by 4 dpf,

with strong signals in the intestinal bulb by 5 dpf

and thereafter It therefore appears that zebra¢sh

PEPT1 expression precedes the functional

matura-tion of the gut, which occurs by 5 dpf and makes the

¢sh ready to perform ¢rst feeding and digestion of

ex-ternal food By 5 dpf, the larva starts the active search

for external food, although a remnant of the yolk is

still present Taken together, these observations

sup-port the notion that the intestinal bulb is the site of

the most e⁄cient absorption of peptides in zebra¢sh,

with PEPT1 being expressed at high levels before the

animal starts to rely autonomously on external food

In summary, zebra¢sh PEPT1 plays a pivotal role in

driving the absorption of dietary di- and tripeptides

Zebra¢sh PEPT1 is adapted to transport at alkaline

pH In the zebra¢sh, PEPT1 is expressed early in the

proximal intestine (e.g the intestinal bulb), starting

at 2 dpf and thus preceding functional maturation of

the gut, ¢rst feeding and complete yolk resorption

Currently, zebra¢sh PEPT1 is fully recognized as a

useful marker to study zebra¢sh gut regionalization,

di¡erentiation and morphogenesis (see e.g Zecchin,

Filippi, Biemar,Tiso, Pauls, Ellertsdottir, Gnˇgge,

Bor-tolussi, Driever & Argenton 2003)

Towards the molecular and functional

description of PEPT1 in other ¢sh species

After the molecular and functional characterization

of the zebra¢sh transporter, the interest in PEPT1 in

teleost ¢sh has grown This increased attention

mainly comes from the observation that teleosts can

e⁄ciently utilize dietary di- and tripeptides for

devel-opment, growth and metabolism and, consequently,

that balanced peptide-based diets or peptide rather

than amino acid supplementation would be highly

bene¢cial in solving the nutritional inadequacy of

formulated feeds for cultured ¢sh (see e.g

Zamboni-no Infante, Cahu & Peres 1997; Dabrowski, Lee &

Rinchard 2003; Aragao, Conceicao, Martins, nestad, Gomes & Dinis 2004; Dabrowski, Terjesen,Zhang, Phang & Lee 2005; Tesser, Terjesen, Zhang,Portella & Dabrowski 2005;Terjesen, Lee, Zhang, Fail-

Rn-la & Dabrowski 2006; Zhang, Dabrowski, Hliwa & mulka 2006; Rnnestad, Kamisaka, Conceicao,Morais & Tonheim 2007; Ostaszewska, Dabrowski,Hliwa, Gomo¤zka & Kwasek 2008) This next section

Go-is intended to provide a brief overview of the currentstate of the art

PEPT1-related expressed nucleotide sequences asfound in various teleost ¢sh species and currentlyavailable in GenBank are summarized in Table 3.With a panel of ¢sh molecular tools available, severalcloning projects have been initiated and are now pro-gressing fast The cloning of a cDNA encoding forAtlantic cod (Gadus morhua) PEPT1 has beenachieved (Rnnestad, Gavaia, Viegas, Verri, Romano,Nilsen, Jordal, Kamisaka & Cancela 2007) and, toour knowledge, there are at least six more cloningprojects underway, aimed at the functional charac-terization of PEPT1 in Antarctic crocodile ice¢sh(M Ma⁄a, pers comm.), Atlantic salmon (I Rnnes-tad, pers comm.), common carp (Cyprinus carpio; T.Ostaszewska & K Dabrowski, pers comm.), rainbowtrout (T Ostaszewska & K Dabrowski, pers comm.),European sea bass (Dicentrarchus labrax; G Terova &

M Saroglia, pers comm.) and China rock¢sh bastes nebulosus; J J Amberg, pers comm.) For twospecies, namelyAtlantic salmon and Antarctic croco-dile ice¢sh, the functional clones are already avail-able and the kinetic characterization in X laevisoocytes is in progress (Table 3) Currently, the onlyfunctional data published in full paper form, besidesthe zebra¢sh, regard the preliminary functionalexpression of a H1/peptide cotransport activity afterinjection into X laevis oocytes of total RNA isolatedfrom Antarctic crocodile ice¢sh intestinal mucosa.Injection of mRNA stimulatesD-Phe-L-Ala uptake in

(Se-a dose-dependent m(Se-anner (Se-and (Se-an excess of Gly-L-Glnsigni¢cantly inhibits D-Phe-L-Ala transport (Ma⁄a

et al 2003)

The rest of the published data on PEPT1-type tide transporters in ¢sh contribute towards under-standing the tissue distribution of PEPT1 in thespecies analysed, namely Atlantic cod, Orientalweatherloach (Misgurnus anguillicaudatus) and or-ange-spotted grouper (Epinephelus coioides)

pep-The Atlantic cod is a key commercial species inmany North Atlantic countries that has recentlybeen targeted for aquaculture, mainly due to deple-tion of natural stocks by over¢shing (see e.g BranderDi- and tripeptide transport in ¢sh gut T.Verri et al Aquaculture Research, 2010, 41, 641^653

r 2009 Blackwell Munksgaard

2007) A full-length cDNA that encodes for the

Atlan-tic cod PEPT1-type peptide transporter has been

cloned (Rnnestad, Gavaia et al 2007) This cDNA

(codPepT1; GenBank Acc No AY921634) is 2838 bp

long, with an open reading frame of 2190 bp

encod-ing a putative protein of 729 amino acids that shares

63% identity with the zebra¢sh PEPT1 As for

zebra-¢sh, hydropathy analysis suggests at least 12

poten-tial TMDs and a large extracellular loop between

TMDs IX and X Unfortunately, when injected into X

laevis oocytes, codPepT1 cRNA did not drive

bio-synthesis of any functional protein (I Rnnestad,

pers comm.) Therefore, no functional analysis of the

Atlantic cod PEPT1is currently available (seeTable 3)

In adult Atlantic cod, PEPT1 mRNA is highly

ex-pressed in the intestine and well exex-pressed in both

the kidney and the spleen (a very slight RT-PCR signal

is also detectable in the ovary; Rnnestad, Gavaia

et al 2007) This expression pattern is similar to that

of zebra¢sh PEPT1 A more detailed analysis of the

regional distribution along the intestinal tract

indi-cates that Atlantic cod PEPT1 is ubiquitously

ex-pressed in all segments beyond the stomach,including the pyloric ceca (Rnnestad, Gavaia et al

2007 It exhibits a lower expression only in the mostposterior portion of the intestine This ¢nding sug-gests that Atlantic cod may have a very high capacity

to absorb small peptides from dietary protein tion, with absorption occurring in most parts of thepost-gastric canal The low expression in the last seg-ment that includes the hindgut indicates that thissegment is not, or only slightly, involved in peptideabsorption In larval Atlantic cod, a detailed analysis

diges-of the regional distribution along the gut (based onquantitative RT-PCR and in situ hybridization studies)has revealed that PEPT1 mRNA is ubiquitously ex-pressed in the epithelium of all segments posterior

to the oesophagus, with the only exception of thesphincter regions (Amberg, Myr, Kamisaka, Jordal,Rust, Hardy, Koedijk & Rnnestad 2008) This spatialexpression pattern is observed at hatching and ismaintained in the more developed gut (22 dayspost hatching) Temporal analysis of expression inlarval Atlantic cod gut indicates that PEPT1 mRNA

Table 3 PEPtide transporter 1 (PEPT1)-related expressed nucleotide sequences in various teleost ¢sh species as available in GenBank (annotated sequences only)

Description Species

GenBank Acc No.

Functional analysis References

mRNA, complete cds Zebrafish (Danio rerio) AY300011 Yes Verri et al (2003)

Antarctic crocodile icefish (Chionodraco

hamatus)

Not yet released Yes M Maffia, pers comm.

Atlantic salmon (Salmo salar) Not yet released Yes I Rønnestad, pers comm Atlantic cod (Gadus morhua) AY921634 No Rønnestad, Gavaia et al (2007) China rockfish (Sebastes nebulosus) EU160494 No GenBank submission

European sea bass (Dicentrarchus labrax) FJ237043 No GenBank submissionw

mRNA, partial cds Antarctic crocodile icefish (Chionodraco

hamatus)

AY170828 – Maffia et al (2003)

Oriental weatherloach (Misgurnus

anguillicaudatus)

DQ668370 – Gonc¸alves et al (2007)

Common carp (Cyprinus carpio) EU328390 – GenBank submissionz

Rainbow trout (Oncorhynchus mykiss) EU853718 – GenBank submission‰

European sea bass (Dicentrarchus labrax) AM419037 – GenBank submissionz

EST, expressed

sequence tags

Channel catfish (Ictalurus punctatus) CK416736 – GenBank submissionk

Amberg J.J., Anderson C.L., Hill R.A., Rust M.B & Hardy R.W Aquaculture Research Institute, University of Idaho, Hagerman, ID, USA wTerova G., Rimoldi S., Cora S., Bernardini G., Gornati R & Saroglia M Department of Biotechnology and Molecular Sciences, Univer- sity of Insubria, Varese, Italy.

zNie G.X., Zheng J.L & Song D.Y Life Sciences, Henan Normal University, Xinxiang, Henan, China.

‰Ostaszewska T., Szatkowska I., Muszynska M., Grochowski P & Dabrowski K Department of Ichtyobiology and Fisheries, Warsaw Agricultural University, Warsaw, Poland.

zHakim Y Animal Science, Hebrew University Jerusalem, Rehovot, Israel.

kLiu Z.J The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Auburn University, Auburn, AL, USA.

cds, coding sequence.