Novel methodologies in marine fish larval nutrition

Novel methodologies in marine fish larval nutrition

Luis E. C. Conceicao • Claudia Aragao •

Nadege Richard • Sofia Engrola • Paulo Gavaia •

Sara Mira • Jorge Dias

Received: 2 October 2009 / Accepted: 10 December 2009 / Published online: 25 December 2009

� Springer Science+Business Media B.V. 2009

Abstract Major gaps in knowledge on fish larval

nutritional requirements still remain. Small larval

size, and difficulties in acceptance of inert microdiets,

makes progress slow and cumbersome. This lack of

knowledge in fish larval nutritional requirements is

one of the causes of high mortalities and quality

problems commonly observed in marine larviculture.

In recent years, several novel methodologies have

contributed to significant progress in fish larval

nutrition. Others are emerging and are likely to bring

further insight into larval nutritional physiology and

requirements. This paper reviews a range of new tools

and some examples of their present use, as well as

potential future applications in the study of fish larvae

nutrition. Tube-feeding and incorporation into

Artemia of 14C-amino acids and lipids allowed

studying Artemia intake, digestion and absorption

and utilisation of these nutrients. Diet selection by fish

larvae has been studied with diets containing different

natural stable isotope signatures or diets where

different rare metal oxides were added. Mechanistic

modelling has been used as a tool to integrate existing

knowledge and reveal gaps, and also to better

understand results obtained in tracer studies. Popula-

tion genomics may assist in assessing genotype effects

on nutritional requirements, by using progeny testing

in fish reared in the same tanks, and also in identifying

QTLs for larval stages. Functional genomics and

proteomics enable the study of gene and protein

expression under various dietary conditions, and

thereby identify the metabolic pathways which are

affected by a given nutrient. Promising results were

obtained using the metabolic programming concept in

early life to facilitate utilisation of certain nutrients at

later stages. All together, these methodologies have

made decisive contributions, and are expected to do

even more in the near future, to build a knowledge

basis for development of optimised diets and feeding

regimes for different species of larval fish.

Keywords Fish larvae nutrition �Genomics � Proteomics � Tracer studies �Modelling � Metabolic programming

Abbreviations

AA Amino acid(s)

DAH Days after hatching

DIGE Differential in-gel electrophoresis

FA Fatty acid(s)

GC Gas chromatography

HPLC High-performance liquid chromatography

HUFA Highly unsaturated fatty acids

IRMS Isotope-ratio mass spectrometry

MAS Marker-assisted selection

NMR Nuclear magnetic resonance

PAGE Polyacrylamide gel electrophoresis

L. E. C. Conceicao (&) � C. Aragao � N. Richard �S. Engrola � P. Gavaia � S. Mira � J. Dias

CCMAR—Centro de Ciencias do Mar, Universidade do

Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

e-mail: [email protected]

123

Fish Physiol Biochem (2010) 36:1–16

DOI 10.1007/s10695-009-9373-z

PCR Polymerase chain reaction

QTL(s) Quantitative trait loci

SDS Sodium dodecyl sulphate

SNP Single nucleotide polymorphism

SSH Suppression subtractive hybridisation

Introduction

Enhanced production of high quality and healthy fry

is a key target for a successful and competitive

expansion of the aquaculture industry. Despite large

quantities of fish larvae of many species being

already produced, survival rates are often low or

highly variable and growth potential is in most cases

not fully utilised (e.g. Conceicao et al. 2003a; Shields

2001). In addition, quality problems, such as skeletal

deformities (Cahu et al. 2003; Koumoundouros et al.

1997), are still a bottleneck. At least part of these

problems derives from sub-optimal nutrition. In fact,

major gaps in knowledge on fish larval nutritional

requirements, even for the better studied species, still

remain. Very small fish size, and difficulties in

acceptance of inert microdiets, makes progress slow

and difficult. This lack of knowledge in fish larval

nutritional requirements is probably one of the causes

of high mortalities and quality problems commonly

observed in larviculture (Bell et al. 2003; Cahu et al.

2003; Kamler 2008; Takeuchi 2001).

Several novel methodologies, including tracer

studies, genomics, proteomics, systems modelling,

molecular genetics, among others, have contributed

to significant progress in fish larval nutrition in recent

years. These methodologies and others emerging,

such as the use of gnotobiotic systems, are likely to

bring further insight into larval nutritional physiology

and requirements in the years to come. This paper

reviews a range of new tools and some examples of

their present use, as well as potential future applica-

tions, in the study of fish larvae nutrition.

Tracer studies

Tube-feeding

The in vivo method of controlled tube-feeding of

radiolabelled nutrients has been developed by Rust

et al. (1993) and modified by Rønnestad et al.

(2001a), allowing to estimate the metabolic budgets

of nutrients (unabsorbed, catabolised and retained

fractions) in fish larvae. Further refinements of the

method allow studying the dynamics of nutrient

absorption into the larvae, through time-course

experiments and/or by dissecting the larvae in

different compartments, such as gut and body

(reviewed by Conceicao et al. 2007). A detailed

description of the method may be found in Rønnestad

et al. (2001a) and Conceicao et al. (2007). Briefly, the

larvae are tube-fed with 14C-nutrients using a capil-

lary and then transferred to single metabolic cham-

bers containing seawater. At the end of the incubation

period, through the manipulation of the seawater pH,

it is possible to promote the diffusion of the CO2 out

of the seawater and therefore distinguish between the

unabsorbed and the catabolised nutrients.

The method has been applied in studies on

digestion, absorption and metabolic handling of

proteins and lipids in several fish larval species.

Using this methodology, it was shown that fish larvae

absorb faster and retain more efficiently free amino

acids (AA) than protein (Rojas-Garcıa and Rønnestad

2003; Rønnestad et al. 2000). Moreover, it was also

observed that hydrolysed protein is absorbed faster

than intact protein and that absorption of the latter

was dependent on larval age and size (Tonheim et al.

2005). Therefore, these studies reinforced the idea

that fish larvae may have difficulties in digesting diets

based on complex proteins such as fish meal.

Several studies have examined the larval ability to

absorb, retain and catabolise individual dietary free

AA. Studies with different fish species have shown

that larvae preferentially use dispensable AA as an

energy substrate, while indispensable AA are prefer-

entially spared for growth (Appelbaum and Rønnes-

tad 2004; Conceicao et al. 2002; Rønnestad et al.

2001b). Furthermore, metabolic fate of free AA is

species and age dependent: aromatic AA (phenylal-

anine and tyrosine) were preferentially retained

during the accentuated metamorphosis that Senegal-

ese sole (Solea senegalensis) larvae undergoes, while

no clear tendency was observed in gilthead seabream

(Sparus aurata) larvae throughout its smoother

metamorphosis process (Pinto et al. 2009).

The tube-feeding technique has also been used to

assess the effect of AA supplementation at short term

in fish larvae. For instance, Aragao et al. (2004)

2 Fish Physiol Biochem (2010) 36:1–16

123

showed that AA retention increases in larval Sene-

galese sole fed diets with balanced AA profiles.

Moreover, Saavedra et al. (2008a, b) used the tube-

feeding of 14C-labelled AA to investigate the effi-

ciency of dietary AA supplementation and to check

whether several AA were limiting protein synthesis in

white seabream (Diplodus sargus) larvae fed rotifers.

Although the tube-feeding technique has been

mostly used for protein and AA metabolic studies,

this methodology has also been applied to study lipid

and fatty acid (FA) metabolism. Hence, Morais et al.

(2005a, b) showed that dietary protein/lipid ratio

affects FA absorption in Senegalese sole larvae.

Moreover, the chemical form in which the FA is

supplied in the diet (non-esterified, esterified into a

phospholipid or to a triacylglycerol) has shown to

affect its absorption in larval gut (Morais et al. 2005b,

c). Other studies using this technique have demon-

strated that lipid absorption in larval Atlantic halibut

(Hippoglossus hippoglossus) gut decreases with lipid

complexity (Mollan et al. 2008) and that FA digest-

ibility in Senegalese sole larvae increases with

unsaturation degree (Morais et al. 2005b).

As reviewed by Conceicao et al. (2007), the

handling stress imposed to the larvae when using the

tube-feeding technique may alter rates of nutrient

digestion, absorption and utilisation. However, it was

shown that in species resistant to stress as Senegalese

sole, the tube-feeding technique do not affect larval

feeding ability or feed utilisation (Ribeiro et al. 2008)

nor ammonia excretion (Aragao et al. 2004). Never-

theless, care should be taken when analysing the

results obtained with stress-sensitive species. It

should be also kept in mind that tube-feeding

experiments are usually performed in fasted larvae

or in larvae fed a single meal, which may result in

increased absorption efficiency (Conceicao et al.

2007; Engrola et al. 2009a).

The tube-feeding technique may have different

applications and different options are being explored.

For instance, the tube-feeding technique (without

tracers) has been used as a tool to study the regulatory

mechanisms of larval protein digestion (Koven et al.

2002; Rojas-Garcıa and Rønnestad 2002). Recently,

this approach was used in combination with14C-labelled preys (described in the next section) in

order to analyse the relation between cholecystokinin

secretion and larval feed utilisation (Ribeiro et al.

2008). Moreover, as will be described in another

section, this technique may be applied to determine

the bioavailability of individual AA in fish larvae.

Artemia labelling

Live feed labelling has been used in several studies in

order to quantify larval feed intake and to understand

protein and lipid metabolism (Conceicao et al. 1998a;

Govoni et al. 1982; Morais et al. 2006). Particularly,

using a methodology based on radiolabelled Artemia

protein (Morais et al. 2004a) combined with the use

of metabolic chambers (Rønnestad et al. 2001a), as

described in the previous section, it is possible to

determine feed intake, and how the ingested trace

nutrient is digested, retained and catabolised by fish

larvae. The use of such methodology in distinct larval

phases allows an understanding of the larvae diges-

tive development and how larvae are coping at the

metabolic level. Hence, Morais et al. (2004b) and

Engrola et al. (2009a) observed that Senegalese sole

larvae have high Artemia protein absorption/digest-

ibility (57–83% of total Artemia intake), between 8

and 35 days after hatching (DAH). This indicates that

sole have a high capacity for digesting live prey since

young ages. In herring (Clupea harengus) larvae

(31 days after first-feeding), the Artemia absorption/

digestibility was around 60%, with 39% being

catabolised and 20% retained by the larvae (Morais

et al. 2004a).

Protein ingredients, such as fish meal, currently

used in inert diets may be too complex for the

immature digestive system of fish larvae (Conceicao

et al. 2007; Rønnestad and Conceicao 2005), leading to

low protein digestibility. As digestibility and protein

retention are key issues in defining larval growth

performance as well as survival rate, Engrola et al.

(2009b) evaluated the effects of two levels of Artemia

replacement by an inert diet on Senegalese sole, using

tracer methodologies (see Fig. 1). A low Artemia

replacement (20% of total diet) feeding regime had no

effect in Senegalese sole larvae protein utilisation.

However, in sole co-fed a high Artemia replacement

level (58% of total diet) both Artemia protein digest-

ibility and retention efficiency decreased between 6

and 15 DAH (Engrola et al. 2009b). Concerning lipid

utilisation, Mai et al. (2009) observed that Artemia

replacement strategy in Senegalese sole has a toll in

terms of growth and lipid digestibility but does not

seem to compromise lipid metabolic utilisation. Lipid

Fish Physiol Biochem (2010) 36:1–16 3

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retention efficiency before metamorphosis was higher

in co-fed sole, reaching values of 50%, while these

values almost doubled during metamorphosis climax,

ranging up to 80%. This suggests that sole growth

performance is affected during metamorphosis climax

and sole is able to compensate a lower protein

digestibility by increasing lipid retention efficiency.

Therefore, tracer studies combined with different

ingredients and feed types may be used in order to

analyse how larvae cope at the metabolic level and

related it to growth performance. A better knowledge

on the way that larvae digest different proteins and

lipids, and how afterwards the resulting AA and FA

are incorporated into protein or catabolised for

energy, would be instrumental in this endeavour,

and would be a major breakthrough in the develop-

ment of optimised larval inert diets.

Bioavailability

In the last few years, methods have been developed to

study the qualitative AA requirements of fish larvae

considering the differences in bioavailability of

individual AA. These methods allow the simulta-

neous estimation of the relative bioavailability of

several individual AA (i.e. a combined measure of

absorption efficiency and rate of catabolism for each

AA when compared with other AA) in fish larvae

(Conceicao et al. 2003a). The basic principle is to

feed larvae with a diet labelled with stable or

radioactive isotopes and then to analyse the relative

isotopic activity for each AA in the larvae and in the

diet.

Conceicao et al. (2003b) estimated the relative

bioavailability of several AA in gilthead seabream

larvae, through a method that combines the use of13C-labelled rotifers and 13C-NMR spectroscopy. This

estimation was also done in sharpsnout seabream

(Diplodus puntazzo) larvae, using 15N-labelled rotifers

and GC-C-IRMS (Saavedra et al. 2007). This last

method is more sensitive than the former and the

production of 15N-labelled microalgae to enrich

the rotifers is much simpler than the production of13C-labelled microalgae (Conceicao et al. 2007).

A new method to analyse the bioavailability of

individual AA is currently being developed, which

will allow determinations with increased accuracy

compared with previous studies. This method com-

bines the use of the in vivo method of controlled

tube-feeding of 14C-labelled AA (described in a

previous section) with a HPLC equipped with a

fraction collector. The combination of these results

allows calculating the specific activity for each AA in

larvae, and the relative bioavailabilities (as described

in Conceicao et al. 2003b) can be estimated after

determining specific activities for each AA in the

tube-fed mixture using the same methodology.

Studies by Conceicao et al. (2003b) and Saavedra

et al. (2007) have shown significant differences in

relative bioavailability of individual AA in both

gilthead seabream and sharpsnout seabream larvae.

Estimation of the relative bioavailabilities of individ-

ual AA can then be used to correct the larval

indispensable AA profile, enabling an estimation of

the ideal dietary AA profile. For instance, applying the

relative bioavailabilities of individual AA obtained by

Conceicao et al. (2003b) to the AA profiles of gilthead

seabream larvae, Aragao Teixeira (2004) showed that

threonine was deficient in diet whereas lysine was not,

contrary to the results suggested without this correc-

tion (Fig. 2). Moreover, Saavedra et al. (2007) showed

that when the bioavailability data was applied to the

AA profiles of sharpsnout seabream larvae, a dietary

deficiency of lysine, methionine and tyrosine was

Fig. 1 Artemia digestibility (% of radiolabel in the sole and

metabolic trap in relation to total radiolabel fed), protein

retention (% of radiolabel in the sole in relation to digested

label) and catabolism (% of radiolabel in the metabolic trap in

relation to digested label) in sole at 6, 15 and 21 days after

hatching (DAH), after 24 h of incubation. Fish were fed with

live Artemia. Values are means ± SD of sole protein

digestibility (n = 9–15). Different letters indicate statistical

differences (P \ 0.05, Tukey’s test) between sole from

different ages (Based on data from Engrola et al. 2009b)


123

evident at certain developmental stages, contrary to

what was apparent without taking into account the

bioavailability data. This methodology provides a

more robust indicator of larval qualitative AA require-

ments than the use of larval indispensable AA profiles.

However, it should be noted that relative bioavaila-

bilities of AA are species specific and are likely to

change throughout fish ontogenesis, due to changes in

type and capacity of the gut transporters and in

activities of enzymes involved in AA catabolism

(Conceicao et al. 2003a).

Protein turnover

The utilisation of absorbed AA will depend not only

on usage of AA for energy purposes, but also on

cycling of tissue proteins, i.e. the net balance of the

rates of protein synthesis and protein turnover. Protein

deposition (or growth) is dependent on this balance,

and may result from an increase in the rate of protein

synthesis and/or from a lower rate of protein turnover

(Wiesner and Zak 1991). Furthermore, higher protein

synthesis rates tend to reduce AA catabolism, as less

free AA will be available as substrates. On the other

hand, the synthesis of proteins has a high energy cost

(Houlihan 1991), and increased protein synthesis

means an augmented demand for energy substrates.

If indispensable AA are used, then the efficiency of

protein utilisation may be reduced.

Fast growing fish larvae might be more efficient in

depositing protein compared to slower growing

larvae (Conceicao et al. 2008). Protein turnover does

not seem to increase concurrently with protein

synthesis as observed in adult fish and mammals

(Houlihan et al. 1994). This may have resulted from a

strong selective pressure for fast growth, and thereby

in efficiency of protein deposition, in fish larvae

(Conceicao et al. 1997a), as it is generally accepted

that larger size is a major selective advantage. Fish

larvae seem to have found two strategies to increase

efficiency of protein deposition: to decrease the rate

of protein turnover or to reduce the costs of protein

synthesis (Kiørboe 1989; Kiørboe et al. 1987).

African catfish (Clarias gariepinus) yolk-sac larvae

growing over 100% day-1 had a protein turnover

comparable to that of other fast growing fish larvae

and juveniles, but protein synthesis costs were found

to be close to theoretical minima (Conceicao et al.

1997a). On the other hand, slower growing first-

feeding turbot (Scophthalmus maximus) larvae have a

very low protein turnover (Conceicao et al. 1997b).

It has also been proposed that once protein

turnover is energy costly, there may be a trade-off

between fast growth and stress-resistance/survival in

fish larvae (Conceicao et al. 2001). A high protein

turnover increases metabolic plasticity, what favours

a rapid adaptation to environmental changes or stress

(Hawkins 1991; Kiørboe et al. 1987). Protein turn-

over in fish increases with environmental stress

(Houlihan et al. 1994; Wilson et al. 1996) and blue

mussel (Mytilus edulis) individuals with higher

protein turnover showed a faster metabolic adaptation

to temperature shock (Hawkins et al. 1987). First-

feeding turbot larvae fed rotifers enriched with an

immunostimulant had a threefold higher protein

turnover when compared to a control group (Con-

ceicao et al. 2001). This may result in larvae with

higher viability and survival in case of environmen-

tal/disease stress. However, as protein turnover is

energy costly, any increase tends to bring a decrease

in growth performance.

It will be interesting to verify in future studies to

what extent this trade-off between fast growth and

stress-resistance/survival applies in fish larvae. In

particular, to study under which circumstances, and in

particular nutritional conditions, fast growth and

increased protein turnover may also be compatible

in fish larvae. This may happen when the necessary

Fig. 2 Comparison of the indispensable amino acid profile of

rotifers with the indispensable amino acid profile of gilthead

sea bream (Sparus aurata) larvae (16 days after hatching)

including (inverted filled triangle) or not (open triangle) the

relative amino acid bioavailabilities (Based on data from

Aragao Teixeira 2004)


123

increase in metabolic rate is compensated by an

optimised nutrient intake.

Diet selection

Currently, Artemia replacement diets are a feeding

strategy widely used in marine fish larviculture since

inert diets are easier to use and have a stable

composition, while composition of live feed can vary

according to culture/enrichment conditions. In addi-

tion, the incorporation of a label in small particles of

larval inert diets, in order to determine larval diet

selection (i.e. feed intake) and nutrient utilisation, is

technically difficult, and protein and AA leaching

problems can easily occur (Kvale et al. 2006; Lopez-

Alvarado et al. 1994; Nordgreen et al. 2007; Yufera

et al. 2002). Therefore, new methodologies need to be

used in order to determine larvae diet selection (live

prey or inert diet) and how feed is being metabolised

by the fish.

In general, diet selection in fish larvae is deter-

mined by visual counting of ingested prey (Haylor

1993; MacKenzie et al. 1999), or particles of inert

diet (Fernandez-Dıaz et al. 1994; Yufera et al. 1995).

This makes estimation of feed intake quite time

consuming and often inaccurate. Therefore, tools

using tracer nutrients have been proposed to deter-

mine the impact of feeding regimes in fish larvae (see

review by Conceicao et al. 2007). The use of stable

(e.g. 13C or 15N) or radio tracer labelling molecules

(e.g. 3H, 14C or 35S) has improved and simplified the

quantification of feed intake and nutrient utilisation

(Boehlert and Yoklavich 1984; Conceicao et al. 2001;

Engrola et al. 2009a; Gamboa-Delgado et al. 2008;

Jomori et al. 2008; Kolkovski et al. 1997; Kvale et al.

2006; Rust 1995). In Senegalese sole fed with an

Artemia replacement strategy, it was possible to

observe that 16–17 DAH postlarvae presented an

Artemia intake of 11% and an Artemia protein

digestibility of 57% (Engrola et al. 2009a) and that

the contribution of Artemia to tissue growth was 62%

(Gamboa-Delgado et al. 2008). Using a double-

isotope labelling method, 14C-labelled inert diet and3H-labelled Artemia, it was possible to observe that

gilthead seabream larvae increase the feed intake of

inert diet in the presence of Artemia (Kolkovski et al.

1997). The methods that use radio tracer molecules

also have some drawbacks; the feed intake quantifi-

cation is measured in small volumes quite different

from standard larvae rearing conditions and produces

radioactive waste. On the other hand, the feed intake

methods that use stable isotopes can be applied in

standard rearing conditions, but can be expensive

when diet enrichment is required.

Inert markers (e.g. yttrium oxide, cholestane or

chromic oxide) are widely used in juvenile and adult

fish nutrient utilisation studies (Austreng et al. 2000;

Carter et al. 2003). In fish larvae, nutrient digestibility

was not usually determined with such methodologies,

due to technical constraints in feed manufacture.

However, recent improvements in feed manufacture

technologies and analytical methods allowed the

incorporation of inert markers in larval feeds (Cook

et al. 2008). Consequently, Johnson et al. (2009) were

able to incorporate yttrium oxide in inert diet and

Artemia and to assess that 58 DAH Atlantic cod

(Gadus morhua) larvae had a feed intake between

0.57–0.79% and 0.67–0.84% for the inert diet and

Artemia, respectively. However, this method is quite

time consuming, and its application is species

specific, depending on the possibility of implement-

ing a faecal collection procedure. Other methodolo-

gies, such as the use of alginate-based inert diet,

which is possible to extract and measure chlorophyll,

was a step forward in the direct assessment of larvae

inert diet intake (Kelly et al. 2000). This method may

help researchers and commercial companies to access

feed utilisation in large-scale facilities.

Therefore, several methods may be used for

studying larvae diet selection. However, at the

moment, none presents the perfect combination of

being simple and easy to perform routinely.

Mechanistic modelling

Mechanistic modelling is a holistic approach that

integrates knowledge on growth and metabolism and

allows identifying gaps in knowledge (e.g. Baldwin

and Sainz 1995; Conceicao et al. 1998b; Gill et al.

1989). A dynamic mechanistic model simulates

physiological processes in time, based on the under-

lying biochemical mechanisms. Model parameters

have as much as possible a biological/biochemical

meaning. Such models are made of a set of equations

that calculate the transfer of nutrients (e.g. AA, FA)

between pools considered (e.g. liver lipids, muscle

proteins). A mechanistic model is always a trade-off


123

between an accurate representation of reality, and the

knowledge available to define equations and quantify

their parameters. Therefore, compromises are neces-

sary, and every model is a simplification of reality,

where usually only the main processes are consid-

ered. Once the model is tested, improvements can be

made, so modelling may be seen as a continuous

process of experimentation and mathematical

formulation.

Conceicao et al. (1993, 1998b) developed a

mechanistic model that simulate growth and bio-

chemical composition during fish larval ontogeny.

The objectives of this model was to contribute to a

better understanding of growth, nutritional require-

ments and the underlying metabolic processes, in

order to contribute to better feeding regimes for fish

larvae. The model is driven by feed intake, with

absorbed nutrients being used either for energy

production or biosynthesis, based on the stoichiom-

etry of intermediary metabolism. Based on model

simulations, Conceicao et al. (1998b) suggest that if

the dietary AA profile is imbalanced in periods of

high feed consumption, high AA losses and a high

lipid deposition will occur. Simulations with this

same model also suggest that high dietary lipid

content will result mostly in lipid deposition and also

a small protein-sparing effect.

Modelling may also be used to assist in the

interpretation of the information from tracer studies,

through the study of the nutrient flow kinetics. A

dynamic mechanistic model that simulates the AA

metabolism of Senegalese sole larvae based on a

previous work with tracer studies has been proposed

(Conceicao and Rønnestad 2004). This model aimed

to improve the understanding of larval digestion and

absorption of dietary AA and postprandial AA

metabolism. The model is driven by AA intake, and

the absorbed dietary AA are either deposited as

protein or used in energy production. The model

output suggests that the AA content of the feed has a

major contribution to the composition of the free AA

pool. Furthermore, the rates of protein synthesis and

AA catabolism seem to increase rapidly after the

meal, with postprandial metabolism peaking only 1 h

after the meal, and returning to baseline values 2 h

later. Together, this suggests a rapid handling of the

Artemia protein by sole larvae, and that full use of the

growth potential of sole and other fish larvae requires

feeding at a high frequency.

Future use of modelling techniques may consider-

ably accelerate the development of optimised diets

and feeding strategies for several fish larvae species.

Its major contribution will likely be to identify gaps

in knowledge, and in particular in evaluating the

relative importance of different factors, nutritional,

environmental and zootechnical, in larval

performance.

Population genomics

It is commonly accepted that some particular traits

are inherited by progeny. Thus, tracing individuals to

their ancestry is a useful approach when evaluating

their performance. Considering a particular trait of

interest such as growth rate, assigning individuals to

families and identifying the crosses which confer a

better performance of progeny is particularly

interesting.

Molecular markers allow inferring paternity and

tracing individuals to their origin, and microsatellites

are the marker of choice for paternity analysis,

mainly due to their high levels of variability (Liu and

Cordes 2004). Few as 4–10 microsatellite loci can be

used to assign more than 90% of individuals with a

95% confidence level to single pair of parents

(Bekkevold et al. 2002; Blonk et al. 2009; Brown

et al. 2005), although the number of loci depend on

their variability and on the size of the breeding

population. Some caution should also be considered

when using microsatellites since high frequencies of

genotyping error such as null and drop out alleles are

frequently reported (Bonin et al. 2004; Castro et al.

2004, 2006) and such errors may bias the analyses.

Also monitoring genetic diversity and relatedness

between individuals selected as breeders is important

to avoid mating among close relatives and preventing

genetic erosion of progeny stocks and the negative

consequences of high levels of inbreeding (Gallardo

et al. 2004; Porta et al. 2006). Thus, molecular

markers may be used on a routine basis to monitor

genetic diversity as it was applied to gilthead

seabream (Blanco et al. 2007; Borrell et al. 2007),

trout (Oncorhynchus mykiss, Gross et al. 2007; Salmo

trutta, Was and Wenne 2002), Atlantic salmon

(Salmo salar, Norris et al. 2000), Atlantic cod

(Pampoulie et al. 2006) and Japanese flounder

(Paralichthys olivaceus, Liu et al. 2005).


123

Selective breeding programmes can be established

based on a specific trait, by retaining the most

interesting individuals to constitute the broodstock

population. However, evaluating the parental contri-

bution prior to the selective programme might be a

useful step in order to identify the breeders with

higher contribution to the progeny. The programme

can then select among those, targeting the traits of

interest, which will increase the probability of traits

inheritance by their offsprings, raising the success-

fulness of the selection programme. Usually, selec-

tive breeding programmes have targeted traits that

may be easily individually recorded and improved

using mass selection (body weight, growth, etc.).

However, traits that are difficult or express late in the

life of the organism such as feed efficiency or

condition factor, may be considered as good candi-

dates to perform marker-assisted selection (MAS;

Chistiakov et al. 2006). As a prerequisite for MAS,

there must be a known association between genetic

markers and genes affecting the phenotype (trait) of

interest. Searching for those associations corresponds

to search for quantitative trait loci (QTL; Liu and

Cordes 2004; Massault et al. 2008). Several QTLs

have been identified in fish related to body weight

(O’Malley et al. 2003; Reid et al. 2007) or body

length (Borrell et al. 2004).

Another way of searching for variation among

individuals linked to traits of interest is the candidate

gene approach which consists in looking for variation

at genes with a known role in the physiology and

explains the phenotypic variability found. Ten can-

didate genes related to the growth hormone axis have

been studied for growth related traits in Arctic charr

(Salvelinus alpinus) and one single nucleotide poly-

morphism (SNP) was found to be associated with

growth (Tao and Boulding 2003). Suppression sub-

tractive hybridisation (SSH) is currently the method

frequently used to identify genes differentially

expressed between individuals that might point out

new candidate genes.

Molecular markers have proved to be a useful tool

to be implemented in breeding programmes to

identify and trace larval fish to their ancestry.

However, looking for particular traits of interest

namely those related to growth and nutrient utilisa-

tion in fish, and larvae in particular, that allows the

construction of QTL maps is still at the very early

stages.

Combining resources of populations and genomics

will allow the use of population-based studies to

detect specific variation affecting complex traits

making possible in a near future the implementation

of MAS in several aquaculture species. Applying

these methodologies as early as the larval stage may

speed up selection progress and bring clues for

optimisation of nutrition and growth. Progeny testing

may also be instrumental in reducing or at least

controlling genetic variability in nutritional studies.

Functional genomics

The increasing amount of knowledge being generated

by gene cloning and from the genome and transcrip-

tome sequencing programs for different aquaculture

produced fish species, allows researchers and pro-

ducers to have a wider view on how nutritional

factors influence gene expression and regulation.

The already completed zebrafish (Danio rerio)

genome allowed the generation of new tools such as

microarrays that make possible to test the effects of a

given experimental condition on the overall gene

expression of a given species or developmental stage,

and together with quantitative approaches by real-

time PCR give a clear view on the alterations

occurring in a set of selected genes. An Atlantic

salmon 16 K cDNA array developed by the GRASP

consortiums (von Schalburg et al. 2005) and a new

gene expression microarray recently developed for

the gilthead seabream comprising almost 20000

clusters (Ferraresso et al. 2008) will allow researchers

to follow the alterations in gene expression that occur

during larval development or the ones induced by

nutritional trials. In addition to microarrays that are

hybridization-based techniques, recent approaches

using large-scale mRNA sequencing are giving new

insights into gene expression profiles (Cloonan et al.

2008; Tang et al. 2009). In recent years, with the

growing number of molecular tools available, the

global analysis of the transcriptome has increased its

importance on understanding how the alteration of

gene expression contributes to metabolic processes,

biological pathways and molecular mechanisms that

regulate cell fate, development and diseases.

The molecular cloning of important digestive

enzymes in fish, such as the gastric pepsinogen

(Darias et al. 2005), the pancreatic enzymes trypsin


123

(Male et al. 2004), amylase (Darias et al. 2006;

Douglas et al. 2000), bile salt-activated lipase (Perez-

Casanova et al. 2004), phospholipase A2 (Zambonino

Infante and Cahu 1999) or the intestinal enzymes

leucine-alanine peptidase, aminopeptidase or alkaline

phosphatase enable a better understanding of the

molecular events complementing the traditional

determination of enzyme activity levels in fish larvae

studies. By combining a quantitative real-time PCR

approach together with the localisation by in situ

hybridisation of the cell populations responsible for

active gene expression at a given time or condition, it

is possible to have an integrated response of how an

aquaculture produced species can physiologically

process the components of the nutritional formula-

tions offered as feed. Moreover, when a given

nutrient is tested, it is possible to study the presence

of the enzymes responsible for its degradation and in

this way a more adequate diet formulation adapted to

each developmental stage may be produced. Exam-

ples of metabolic enzymatic modulation in response

to diet changes are the studies of Seiliez et al. (2003),

Izquierdo et al. (2008) and Vagner et al. (2009),

reporting adaptations of delta 6 desaturase gene

expression to dietary FA composition changes in

marine larvae (see Fig. 3) and identifying some

factors involved in this regulatory mechanism (Vag-

ner et al. 2009). Further, changes in digestive enzyme

expression when the diet shifts from carnivore to

herbivore were recently shown for thicklip grey

mullet (Chelon labrosus) larvae (Zouiten et al. 2008),

suggesting that digestive enzyme activity is

genetically programmed to match the developmental

alterations in the diet. Modulation of dietary lipids

was shown to influence the gene expression of

unsuspected enzymes such as gluconeogenic

enzymes in rainbow trout (O. mykiss), without

affecting other digestive or hepatic enzymes (Ducas-

se-Cabanot et al. 2007). Moreover, vitamin A, an

essential nutrient involved in multiple molecular

mechanisms and with great influence in early larval

stages, has been shown to affect greatly the early

development of European sea bass (Dicentrarchus

labrax) and Japanese flounder (Dedi et al. 1997;

Villeneuve et al. 2004, 2005, 2006). Villeneuve et al.

(2004) have identified the different forms and the

tissue pattern of expression of RA receptors and

demonstrated that their expression levels are affected

by modulating dietary RA levels (Villeneuve et al.

2005). In both species, hypervitaminosis A causes

severe morphogenic alterations affecting in particular

skeletal formation (Dedi et al. 1997; Villeneuve et al.

2006).

These molecular approaches allow the identifica-

tion of new target genes/proteins involved in nutrient

metabolism and a better prediction of a species

response to different diets. It can also be expected

that in a near future, high-throughput genomic/

transcriptomic applications combined with more

detailed quantification methods will be crucial for a

detailed understanding of the metabolic pathways and

the effects of nutritional factors.

Functional proteomics

As an end-product of gene expression, the study of

protein expression allows one to view any functional

and/or structural effect caused by an environmental

modification on the organism. Two-dimensional

electrophoresis, which combines the separation of

the proteins according both to their isoelectric point

(by isoelectric focusing) and their molecular weight

(by SDS–PAGE) (Lopez 2007), followed by identi-

fication of the proteins of interest by mass spectrom-

etry (Canas et al. 2006) is the most common strategy

used nowadays for proteome analysis.

Proteomics allows the detection of all the proteins

present in a tissue or a cell and the assessment of

changes in their relative abundance in response to a

given environmental modification, at a given time

Fig. 3 Average D6 desaturase-like gene expression by real-

time PCR in gilthead sea bream (Sparus aurata) larvae fed

microdiets formulated with different oils. B-actin was used as

reference. Results are standardised in relation to expression of

fish fed the microdiet with fish oil (Based on data from

Izquierdo et al. 2008)


123

point. This technology enables thus to expand the

experimental focus from targeted proteins to a whole

range of proteins at the same time, without any

‘‘a priori’’ knowledge and provides also some infor-

mation about the regulation level of gene expression,

such as potential post-translational modifications (e.g.

glycosylation, phosphorylation, acetylation, ubiquiti-

nation), thus being complementary to genomic

studies.

The use of proteomics in fish research is at a

relatively early stage compared to terrestrial verte-

brates. It has already been applied in the field of

nutrition, enabling to point out metabolic changes

occurring in response to dietary manipulations such as

a variation in energy content or the incorporation of

plant protein sources in rainbow trout diet (Kolditz

et al. 2008; Martin et al. 2003; Vilhelmsson et al. 2004).

Concerning the larval stage, the application of the

proteomic approach is more challenging due to the

small size of the individuals and thereby the consid-

erable amount of material needed for such an

analysis, especially when the focus is a specific

tissue. Until now, few proteomic studies conducted

on fish larvae are available and those that are, focused

mainly on changes of proteome expression during

fish development (Focant et al. 2003; Link et al.

2006a; Sveinsdottir et al. 2008). Indeed, to our

knowledge, no study has yet been applied the

proteomic approach to assess the effect of dietary

manipulations on fish larvae metabolism. Ongoing

studies on this respect are integrating comparative

proteome analysis in determining the impact of

dietary nutrients such as vitamin K (Richard et al.

2008) or protein hydrolysates (Richard et al. unpub-

lished data) on marine fish larvae metabolism. Albeit

the application of this proteomic approach in fish

larvae is to this date not widely used due to the

difficulties mentioned earlier, recent technical

advances in this field such as DIGE (differential in-

gel electrophoresis) technology, which shows a

higher detection sensitivity that reduces the amount

of tissue needed for performing a comparative

proteome analysis (Marouga et al. 2005), combined

with new microdissection techniques, are promising

to overcome these obstacles. Moreover, DIGE tech-

nology has already been used in developmental

biology studies conducted in fish (Damodaran et al.

2006; De Wit et al. 2008), as early as the embryo

stage (Link et al. 2006a, b).

Metabolic programming

Knowledge from mammalian models demonstrate

that prenatal or early postnatal events (e.g. maternal

nutrition, maternal vaccination, abiotic factors),

exerted at critical developmental windows, result in

lifelong contributions to postnatal growth potential

and health status (Lucas 1998). This concept, known

as metabolic programming (see Fig. 4), has been

studied in rodent models in association to diseases

such as the metabolic syndrome or diabetes (Water-

land and Garza 1999). Possible biological mecha-

nisms for ‘‘imprinting’’ the nutritional programming

stimulus until adulthood include adaptive changes in

gene expression, preferential clonal selection of

adapted cells in programmed tissues, and pro-

grammed differential proliferation of tissue cell types

(Lucas 1998; Metcalfe and Monaghan 2001).

The potential of nutritional or metabolic program-

ming in fish remains unexplored and has yet to be

clearly validated. In Atlantic salmon, Macqueen et al.

(2008) showed that changes in water temperature

during embryogenesis dictated adult myogenic phe-

notype (fibre number, diameter). In another recent

work by Geurden et al. (2007), it was shown that a

short hyperglucidic stimulus at first feeding, upreg-

ulated carbohydrate digestive enzymes in rainbow

trout at a latter juvenile stage, suggesting some long-

term physiological changes. Additionally, European

Fig. 4 The concept of metabolic programming. Early postna-

tal stimulus (e.g. nutritional, abiotic), exerted at critical

developmental windows, may result in increased heterogeneity

of metabolic responses leading to lifelong contributions on

postnatal growth potential and health status


123

seabass fed a highly unsaturated fatty acid (HUFA)-

deficient diet during an early larval stage showed a

positive modulation of the D6-desaturase transcrip-

tion in juveniles fed a HUFA-deficient diet (Vagner

et al. 2007). The persistency of the observed effects

and the mechanisms underlying such changes is

currently unknown.

An enhancement of the embryo nutritional status,

through optimised maternal nutrition, has clearly

been associated with beneficial effects of offspring,

on both oviparous and placental viviparity animals

(Metcalfe and Monaghan 2001). In farmed fish, the

nutritional value of yolk reserves is conditioned by

the dietary status of broodstock fish and it can be

enhanced through maternal transfer of liposoluble

vitamins, antioxidants and HUFA (Izquierdo et al.

2001). The supplementation of broodstock diets with

such selected nutrients has been related to improved

spawning success and egg quality in both freshwater

and marine species. Once spawned and fertilised the

oviparous fish eggs operate as closed systems; only

respiratory gases, heat and negligible amounts of

solutes and water are exchanged freely, as a result of

an extremely low permeability of the egg surface

membranes (Kamler 2008). A great amount of data

has been gathered in various fish species, regarding

the biochemical composition and the patterns of

nutrient allocation during the yolk-sac stage of larvae

development (Kamler 2008). Typically, fish ontogeny

begins with a short period of carbohydrate use, soon

switched to free amino acids (FAA) as the most

important fuel. Lipid mobilisation follows after

hatching in response of increased energy demand.

At the onset of first-feeding, lipids provide energy for

swimming activity. The second peak of amino acid

catabolism occurs after depletion of endogenous

reserves, when body protein-bound amino acids are

mobilised (e.g. vitellogenin degradation). Timing and

extent of these shifts is species specific and modified

by egg properties (with or without oil globule)

(Kamler 2008; Ronnestad et al. 1999). If not

adequately fed at the onset of exogenous feeding,

marine fish larvae may pass through a status of

deprivation of selected essential nutrients. Despite its

short-duration, a deficiency in essential nutrients

during early development is particularly important,

given the extremely high rates of growth and protein

synthesis. Even if an organism apparently recovers

from this deprivation, the nutritional deficit

experienced during early development can have

pervasive and permanent effects on the adult indi-

viduals (Metcalfe and Monaghan 2001). Despite not

yet evaluated in fish, such undernutrition stimulus

could programme growth potential, metabolic path-

ways and contribute to a later higher size dispersal of

juveniles (fast and slow growers). However, the long-

term persistency of molecular adaptations following

early nutritional stimulus and its interaction with

epigenetic factors (e.g. DNA methylation patterns) in

fish with a long life cycle still needs to be

substantiated.

A sound validation of the nutritional programming

concept in fish will be an important breakthrough

achievement in the area of larvae nutrition. By taking

advantage also of integrative nutrigenomic tools,

future research should contribute towards a better

insight and identification of nutritional triggers that

programme key biological and metabolic processes

occurring in early developmental phases of fish (e.g.

protein retention, muscle fibre myogenesis, skeletal

metabolism). Its practical implications can be wide.

For instance, the identification of a nutritional trigger

to enhance the metabolic utilisation of carbohydrates

in fish could open new avenues for the utilisation of

vegetable ingredients in fish feeds. On a broader

sense, nutritional programming at the broodstock

and/or larval level, associated with feeding and

rearing practices could play an important role in

controlling important economic and quality traits in

aquaculture.

Conclusion

Methodologies such as tracer studies and functional

genomics have already made decisive contributions

to build a better knowledge basis for development of

optimised diets and feeding regimes for different

species of larval fish. Nevertheless, detailed under-

standing of the nutritional physiology and require-

ments of fish larvae, even for the better studied

species, is far from being reached. Other recent

methodologies not under the scope of this review, as

the use of gnotobiotic systems, may allow under-

standing how gut microbial communities may con-

tribute to nutrient processing and absorption in fish

larvae, opening new perspectives for research in

larval nutrition. Therefore, there is still much room


123

for improvement in diet formulation and feeding

regimes for fish larvae, which will be reflected in

better performance and quality. The methodologies

discussed in the present paper will certainly plain a

decisive role in attaining those goals (see Table 1).

Acknowledgments C. Aragao, S. Engrola, S. Mira and N.

Richard acknowledge financial support by Fundacao para a

Ciencia e Tecnologia, Portugal, through grants SFRH/BPD/

37197/2007, SFRH/BPD/49051/2008, SFRH/BPD/23514/2005

and SFRH/BDP/34888/2007, respectively. Project HYDRAA—

PTDC/MAR/71685/2006, granted by ‘‘Fundacao para a Ciencia

e Tecnologia’’ (FCT), Portugal, with the support of FEDER, is

also acknowledged.

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Methodology Contribution

Tracer studies Digestibility of different nutrients; diet preferences; short-term nutritional effects; interaction between

nutrients; screening of most promising graded levels and nutrient combinations

Mechanistic

modelling

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Population

genomics

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Functional

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Functional

proteomics

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Metabolic

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