Effects of nutrition on larval growth and survival in bivalves

Effects of nutrition on larval growth and survival in bivalvesRobert Marshall1,2, Scott McKinley2,3 and Christopher M. Pearce1,4

1 Fisheries and Oceans Canada, Pacific Biological Station, Nanaimo, BC, Canada

2 Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC, Canada

3 Centre for Aquaculture and Environmental Research, University of British Columbia, Vancouver, BC, Canada

4 Fisheries and Aquaculture Department, Vancouver Island University, Nanaimo, BC, Canada

Introduction

Stagnant capture fisheries, a growing population and a

strong demand for aquatic food products have been the

driving forces behind the rapid increase in aquaculture

production since the 1950s (Anonymous 2006). Forecasts

predict that production will need to increase for decades

to come if demands are to be met (Brugere & Ridler

2004). Molluscs account for 23% of current global aqua-

culture production (Anonymous 2006), but procurement

of juveniles (also called ‘seed’ or ‘spat’) for culture pro-

duction may become a bottleneck. Currently, much of the

world’s bivalve aquaculture relies on seed collection from

wild sources (Chavez-Villalba et al. 2002). Natural seed

collection using specialized equipment, such as hanging

spat collectors and cultch bags (Bervera & Monteforte

1995; Tammi et al. 1995; Thorarinsdottir 1995; Garcia

et al. 2003), can facilitate larval settlement and spat

recruitment, but ultimately the success of wild seed collec-

tion is at the mercy of environmental, biological and

anthropogenic factors that are beyond the aquaculturist’s

control (Lubet et al. 1991). Environmental factors such as

phytoplankton abundance (Chıcharo & Chıcharo 2001),

phytoplankton type (Bricelj & MacQuarrie 2007), short-

term weather severity (Strasser et al. 2001) and long-term

climate patterns (Philippart et al. 2003; Valero et al. 2004)

can all impact larval growth and survival and ultimately

limit seed procurement from the wild. Anthropogenic

activities, such as habitat destruction by toxic chemicals,

reduced access to locations used for recreation (Robert &

Gerard 1999) and pressures from the environmental lobby

(Edwards 2005), can also impact the ability of aquacultur-

ists to collect wild seed. All of these factors, which limit

the ability of the farmer to secure reliable and predictable

seed supplies from the wild, can severely handicap devel-

opment of the bivalve aquaculture industry.

To overcome the many problems associated with

wild seed collection there has been a move towards hatch-

ery-reared product. Stabilization of seed supply through

hatchery sources can benefit not only the aquaculture

Correspondence

Robert Marshall, Fisheries and Oceans

Canada, Pacific Biological Station,

3190 Hammond Bay Road, Nanaimo,

BC V9T 6N7, Canada.

Email: [email protected]

Received 17 June 2009; accepted 21 September

2009.

Abstract

This review examines the nutritional factors that influence the growth and sur-

vival of larval bivalves. Factors considered include feed form (live phytoplank-

ton, preserved phytoplankton and artificial feeds) and feed biochemical

composition (protein, lipid, carbohydrate and essential fatty acids). These fac-

tors, as they relate to larval production, are discussed in terms of growth and

survival rates. To facilitate comparisons among larval studies, growth rates and

feeding rates are standardized to common units. In addition, the standardized

results for larvae of the Pacific oyster (Crassostrea gigas Thunberg) are analysed

using linear regression techniques to determine the strength of the correlations

between daily doses of biochemical feed components and daily growth rates.

Piecewise linear spline modelling is used to determine maximum effective dose

response, the point where feeding additional biochemical components to the

larvae yields no significant improvements in growth. Derived from these analy-

ses are suggested daily rations of lipid, protein, carbohydrate, eicosapentanoic

acid, docosahexanoic acid and energy for larvae of C. gigas.

Key words: bivalve, larval diet, larval growth, larval survival, nutrition.

Reviews in Aquaculture (2010) 2, 33–55 doi: 10.1111/j.1753-5131.2010.01022.x

ª 2010 Blackwell Publishing Asia Pty Ltd 33

industry, with predictable and sustainable production, but

also fisheries by reducing the amount of wild seed col-

lected for culture purposes and allowing for the restock-

ing of stressed populations through re-seeding programs

(Beattie 1992; Anonymous 2006). One of the main advan-

tages of hatchery production over wild seed collection is

that conditions in hatcheries can be manipulated to stim-

ulate gonad development and gametogenesis, making it

possible for the production of viable offspring for com-

mercial purposes at virtually any time of the year (Utting

1993). Hatchery rearing of bivalve larvae is not a new

technology and has been practised since at least the 1930s

in North America (Loosanoff & Davis 1952, 1963).

Hatchery production generally consists of two phases, a

gametogenesis or broodstock conditioning period and a

larval rearing stage. Key factors determining success in

both phases include: temperature, salinity, water quality,

nutrition, stocking density and disease (Brenko &

Calabrese 1969; Gallager et al. 1986; Utting 1986; His

et al. 1989; Helm et al. 2004). The focus of this review is

an examination of the effects of various nutritional

factors on larval bivalve development.

Nutrition can be the dominant factor influencing larval

bivalve growth, explaining a greater proportion of the

variability than either temperature or salinity (His et al.

1989). Because of its importance in larval production,

nutrition has been studied for many decades (Loosanoff

& Davis 1963; Webb & Chu 1981; Delaunay et al. 1993;

Martınez-Fernandez et al. 2006). Most of the reserves

stored in eggs during gametogenesis are used up during

embryogenesis (Gallager & Mann 1986; Whyte et al.

1990) so there is a particularly high requirement for

exogenous sources of nutrients during the larval phases

up to settlement (Pernet et al. 2003). Food value is deter-

mined mostly by biochemical composition (lipid, carbo-

hydrate and protein). Lipid, mainly neutral lipid in the

form of triacylglycerol (TAG), has been identified as the

principal source of energy for bivalve larvae in starvation

experiments, followed by protein, with very little energy

contribution from carbohydrate (Millar & Scott 1967;

Holland & Spencer 1973; Gallager et al. 1986; Whyte

et al. 1992). Essential fatty acids (EFAs), particularly the

omega-3 fatty acids eicosapentanoic acid (20:5n-3 (EPA))

and docosahexanoic acid (22:6n-3 (DHA)), are important

to growth and development (Langdon & Waldock 1981;

Webb & Chu 1981) because they are major membrane

components (Dunstan et al. 1994; Hendriks et al. 2003)

and possible modulators of membrane function (Palacios

et al. 2005). In addition, the omega-6 fatty acids

docosapentanoic acid (22:5n-6 (DPA)) and arachidonic

acid (20:4n-6 (AA)) have been identified as potentially

significant to the growth and survival of larval (Pernet

et al. 2005) and post-larval bivalves (Milke et al. 2008).

The capability for synthesis of EFAs in bivalves is very

limited and inadequate to meet their nutritional require-

ments; therefore, they must be supplied exogenously

(Waldock & Holland 1984; Laing et al. 1990; Chu &

Greaves 1991).

A major challenge of reviewing the literature on larval

bivalve rearing is the wide variety of methodologies and

units of measurement used. Despite a relatively long his-

tory of research, there is no standardization of how

results are reported or how feeding rates and stocking

densities are determined. For example, phytoplankton are

usually introduced to larvae at a fixed cell concentration,

generally between 50 000 and 100 000 cells mL)1 (Laing

& Utting 1994; Andersen et al. 2000; Hendriks et al. 2003;

Ponis et al. 2006a), but there is wide variation in larval

stocking densities. Larval densities in studies considered

in this review range from 1 to 23 larvae mL)1 (Robert

et al. 1996; Torkildsen & Magnesen 2004). Moreover, the

concentration of cells presented to the larvae may be

similar, but the rate at which the cells are supplied to the

larval tanks can vary, typically from daily (Utting 1986;

His & Seaman 1992; Taris et al. 2006) to every third day

(Ponis et al. 2003, 2006b). Based on these variations in

experimental methodology, the actual amount of algae

presented on a per-larva basis per unit time can vary

quite substantially among studies. As a result of this

variation, direct comparisons among studies are often

very difficult. Therefore, one of the main objectives of

this review was to standardize the units of both treatment

variables and results from existing literature to help facili-

tate comparisons and interpretations.

Survival and biochemical composition of larvae are

often used as indicators of success, but for the purposes

of this review, growth rates are the key factor considered.

Rapid growth is generally a reliable indicator that culture

conditions are favourable, correlating to higher survival

(Fig. 1 shows an overview of studies on the Pacific oyster

(Crassostrea gigas Thunberg)), earlier settlement (Peche-

nik & Lima 1984; Robert & Gerard 1999) and higher

metamorphic rates (Bricelj & MacQuarrie 2007). Using

standardized growth rates, commercially valuable temper-

ate species of North America and Europe are discussed in

depth. These commercial species represent the largest

proportion of bivalves reported in the scientific literature

and form the main focus of this review. The main species

discussed are: Manila clam (Venerupis (= Ruditapes =

Tapes) philippinarum A. Adams and Reeve) (Soudant

et al. 2004), great scallop (Pecten maximus Linnaeus),

Pacific oyster (C. gigas) and the brooding European flat

oyster (Ostrea edulis Linnaeus). The following sections

discuss the effects of nutrition on larval performance in

terms of algal species, size and digestibility. In addition,

alternative feed sources (preserved algae, formulated

R. Marshall et al.

Reviews in Aquaculture (2010) 2, 33–5534 ª 2010 Blackwell Publishing Asia Pty Ltd

feeds, picoplankton, bacteria and biochemically altered

algae) are reviewed. Rations are discussed and suggestions

made for prescribed levels of particular algal species

relative to their biochemical composition (protein, carbo-

hydrate, lipid and EFAs). To find potential correlations

between the ingested biochemical components of feed

and larval growth rates, standardized results were analy-

sed using linear regression and piecewise spline regression

modelling to determine maximum effective doses. Poten-

tial avenues of future research are also identified.

Methods

Standardization of larval growth rates

Larval growth rates are reported in variable units in the

literature and are in need of standardization so that

studies may be more directly compared. Often, the

results of larval growth experiments are described as a

final size (typically shell length), but the duration of

the experiments may vary, meaning that the actual rate

of growth may not be comparable among studies. To

facilitate comparisons among studies in the current

review, we re-calculated published growth rates and

produced a standardized growth unit (lm day)1). This

unit was calculated by taking the difference between the

initial and final larval length (lm) and dividing it by

the duration of the study (days). If the study reported

growth results in these particular units, they were used

without modification. Studies with a maximum dura-

tion of 16 days were used. The rationale for this was

that the duration from D-stage to metamorphosis for

C. gigas larvae is approximately 14 days under normal

conditions (Rico-Villa et al. 2006). Considering normal

variation in the time required for settlement, 16 days

was accepted as an upper limit. As growth slows

toward metamorphosis (Utting & Spencer 1991), any

studies with prolonged durations (delayed metamorpho-

sis) were not used as these studies would show very

low average growth rates and possibly confound results.

Targeting studies that focus on the earlier phases of lar-

val development, where growth rates are at their maxi-

mum, was not possible because of the low number of

studies that report shell lengths prior to the onset of

metamorphosis.

Standardization of larval feeding rates

For the purposes of this review, the feeding rates of pub-

lished studies were standardized to common units (i.e. cells

larva)1 day)1) to facilitate comparison. These units were

calculated by taking the concentration of algal cells (cells

mL)1) fed to the larvae, dividing by the density of larvae

(larvae mL)1) and dividing by the feeding frequency

(days). Some studies used progressive feeding rates, where

the concentration of algal cells increased as the size of the

larvae increased. In these cases the final cell concentration

was used for the calculation. The actual number of cells

ingested was assumed to be 70% of the feeding rate (Brown

& Robert 2002). Utting (1986), who also fed C. gigas at

various rates, showed that the average ingestion rate of

algae for several trials was 71 ± 23% (mean ± standard

deviation) (calculated from the reported data), with slight

decreases in ingestion rates as the cell concentration

increased. It must be considered that this assumed

ingestion rate is not likely to reflect the actual ingestion rate

for each study. Few studies, however, report actual

ingestion rates and the 70% assumption is the best

available. In all likelihood the relationship between algal

cell concentration and ingestion rate is asymptotic and not

linear as suggested by this assumption. The feeding

rate used in the Brown and Robert (2002) study was

in the upper range of the studies examined (20 000

cells larva)1 day)1 Isochrysis sp., Tahitian strain (T-iso)

(Isochrysis sp. (Tahitian strain) is often wrongly referred to

as Isochrysis galbana (T-iso) or Isochrysis aff. galbana

(T-iso)) and Chaetoceros calcitrans forma pumilum com-

bined) and the cell removal rate was still approximately

70%; therefore, this rate is assumed to be reasonable in the

range of this analysis, but cannot be assumed to be accurate

beyond the ranges examined. Actual ingestion rates

were used in the analysis if this information was reported.

Standardization of the biochemical components ingested

Although standardizing algal feeding rates (by converting

to standardized units of cells larva)1 day)1) is a good

y = 17.881ln(x) + 45.759

R 2 = 0.6342

0

20

40

60

80

100

120

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Figure 1 Relationship between survival rate and growth rate for lar-

vae of Crassostrea gigas. Data represent a compilation of standardized

results from Utting (1986), His et al. (1989), His and Seaman (1992),

Robert et al. (2001), Brown and Robert (2002), Ponis et al. (2003,

2006b,c), Rico-Villa et al. (2006) and Taris et al. (2006).

Nutritional effects in larval bivalves

Reviews in Aquaculture (2010) 2, 33–55ª 2010 Blackwell Publishing Asia Pty Ltd 35

initial step, this process does not reflect potential differ-

ences in the amounts of important cellular biochemical

components that the larvae may be receiving owing to

the use of differing algal species. It is well documented

that phytoplankton species vary in the concentration of

a number of important cellular components, including

protein, carbohydrate, lipid, energy, EPA and DHA

(Strathmann 1967; Webb & Chu 1981; Dunstan et al.

1992, 1994; Thompson et al. 1992; Verity et al. 1992;

Brown et al. 1997). To standardize among studies, we

estimated the amount of these various components that

larvae received per unit time (pg larva)1 day)1). This

was done by first estimating the concentration (pg cell)1)

of protein, carbohydrate, lipid, energy, EPA and DHA in

various algal species, based on results from published

studies (Table 1). Then, we multiplied our standardized

unit for the number of ingested cells (cells larva)1 day)1)

by the estimated level of a biochemical component in a

given algal species (pg cell)1) to provide an estimate of

an ingested biochemical component (pg larva)1 day)1).

To reduce the variability among studies, the analysis was

restricted to one bivalve species, C. gigas, reared within a

relatively narrow temperature range (25–30�C) and a

stocking density of 5–10 larvae mL)1. This species was

selected for the analysis because the bulk of the literature

on larval bivalve growth is related to this particular spe-

cies, allowing for a reasonable sample size. In addition,

studies that used Pavlova species as a feed were excluded

because C. gigas has very low ingestion rates with this

particular algal genus (Ponis et al. 2003; Rico-Villa et al.

2006).

Several biochemical components were not included in

this analysis. The fatty acids AA and DPA were not

included because of a relative lack of research (Pernet

et al. 2005). Individual amino acids were also not consid-

ered because Brown (1991) found that there were only

minor variations in amino acid content among 16 species

of algae commonly used in aquaculture and amino acids

are less likely to be a factor than other nutrients. Vita-

mins were not included in this analysis because of a lack

of research, although one study indicated that some vita-

mins may be limiting at various stages of larval develop-

ment (Seguineau et al. 1996).

Data analysis

Two types of analyses were run on the data to determine

the relationships between various biochemical compo-

nents in the feeds and larval growth rates. Linear regres-

sions were conducted to determine the correlation

between the ingested levels of biochemical factors and lar-

val growth and piecewise linear spline models were run to

determine maximum effective dose levels of the various

biochemical factors. In this case the definition of maxi-

mum effective dose is a modification of that used in

therapeutics (Remmenga et al. 1997), and is the amount

of a chemical component presumed to be ingested above

which there is no significant improvement in growth. It

can also be thought of as the minimum dose required for

maximum growth. The independent variables used in the

analyses were ingested levels of protein, carbohydrate,

lipid, energy, EPA and DHA. The dependent variable was

standardized growth rate. For the regression analysis, the

independent and dependent variables were transformed

with a natural logarithm to linearize and normalize the

data. Inspecting residual plots and applying D’Agostino’s

test for normality confirmed the effectiveness of this

transformation. The linear spline model analysis was

completed on non-transformed data (Remmenga et al.

1997). The linear spline model analysis is a method that

fits two linear lines to a dataset that reaches a plateau and

identifies the intersecting point of the straight lines

beyond which additional nutrients fail to increase growth

rates (Anderson & Nelson 1975). This was accomplished

with NCSS 2007 (NCSS 2007ª, Kaysville, Utah, USA) by

planting a random seed for the intersection and running

a minimum of 100 iterations. This intersection point

identifies the maximum effective dose of the nutrient.

Based on this analysis and the average levels of the bio-

chemical components found in common algal species

used in larval culture, appropriate algal rations were

calculated for optimal growth of larval C. gigas.

The intent of this analysis was to identify correlations

between the nutrient levels presumably ingested by the

larvae and growth rates. It should not be interpreted that

these are necessarily causal effects. A correlation of a

component to high growth rates may simply be the result

of that component correlating positively with another

more influential component. In addition, there may be

interactions between components that are not represented

by this analysis (e.g. vitamins, amino acids, specific fatty

acids) that may confound the results. By conducting this

simplified analysis of data from several sources we can,

however, identify general trends in the data, devise some

general conclusions and identify potential areas for future

research.

Publication bias, known as the ‘file drawer’ effect, in

which negative results are not published is an issue when

analysing data from multiple sources (Rosenthal 1979;

Begg & Berlin 1988; Thornton & Lee 2000). This may

overestimate the effect size (Palmer 1999). Palmer (1999)

suggests that publication bias can be minimized by

including as many unpublished results as possible. This

was not practical in the present review but to try to

minimize this effect, studies with negative controls and

unfavourable results were included.

R. Marshall et al.


Tab

le1

Bio

chem

ical

com

ponen

tsan

ddry

wei

ghts

of

som

eco

mm

only

culture

dphyt

opla

nkt

on

spec

ies

Cla

ssSp

ecie

sPr

ote

in

(pg

cell)

1)�

Car

bohyd

rate

*

(pg

cell)

1)�

Lipid

(pg

cell)

1)�

Ener

gy

(10

)8

cal

cell)

1)�

Dry

wei

ght

(pg

cell)

1)

Tota

lfa

tty

acid

(pg

cell)

1)

EPA

(pg

cell)

1)*

DH

A

(pg

cell)

1)*

Ref

eren

ces

Hap

tophyc

eae

(=Pr

ymnes

iophyc

eae)

Dia

cronem

avl

kian

um

2.5

0.5

45

0.1

8Po

nis

etal

.(2

006c)

Isoch

rysi

sgal

ban

a(T

-iso

)6.8

1.8

5.9

9.3

26

21

3.0

70.0

28

0.2

6N

evej

anet

al.

(2003a)

0.0

28

0.8

2Riv

ero-R

odrigues

etal

.(2

007)

0.0

10.0

9D

elau

ney

etal

.(1

993)

16

0.0

13

0.2

5Pa

tilet

al.

(2007)

0.0

50.8

3M

artinez

-Fer

nan

dez

etal

.(2

006)

Pavl

ova

luth

eri

5.3

0.6

212.3

14.1

95

0.7

70.1

80.0

8D

elau

ney

etal

.(1

993)

Pseu

dois

och

rysi

spar

adoxa

1.6

40.2

30.3

Ponis

etal

.(2

006c)

Bac

illar

iophyc

eae

Chae

toce

ros

calc

itra

ns

0.2

20.0

2Riv

ero-R

odrigues

etal

.(2

007)

C.

calc

itra

ns

2.4

50.4

0.0

3D

elau

ney

etal

.(1

993)

C.

calc

itra

ns

form

apum

ilum

26.9

%TF

A1.7

%TF

ARic

o-V

illa

etal

.(2

006)

C.

calc

itra

ns

(unsp

ecifi

ed

stra

in)

3.8

0.6

81.8

3.6

74

11.3

0.1

70.0

2La

vens

&So

rgel

oos

(eds)

(1996)

8.4

7La

ing

etal

.(1

990)

Chae

toce

ros

gra

cilis

92

5.2

9.7

38

10%

TFA

1.2

5%

TFA

Mar

tinez

etal

.(1

992)

Chae

toce

ros

muel

leri

23.1

50.2

40.0

1M

atin

ez-F

ernen

dez

etal

.(2

006)

Chae

toce

ros

neo

gra

cile

70

12.9

31.4

50.1

6N

evej

anet

al.

(2003a)

Phae

odac

tylu

mtr

icorn

utu

m23

6.4

10.7

22.9

68

76.7

Lave

ns

&So

rgel

oos

(eds)

(1996)

3.7

90.1

5Riv

ero-R

odrigues

etal

.(2

007)

Skel

etonem

aco

stat

um

13.1

2.4

511.5

55

5.6

1.3

80.1

1D

unst

an(1

994)

Pras

inophyc

eae

Tetr

asel

mis

suec

ica

52.1

20.2

16.8

47.3

17

168.2

Riv

ero-R

odrigues

etal

.(2

007)

15.7

Del

auney

etal

.(1

993)

1.3

20

Riv

ero-R

odrigues

etal

.(2

007)

0.3

0,

0.5

20

Utt

ing

(1993)

246.8

22.8

%TF

A,

5.7

%TF

A

0.1

%TF

A,

0.6

%TF

A

Lain

get

al.

(1990)

Chlo

rophyc

eae

Dunal

iella

tert

iole

cta

20

12.2

15

27.9

810

ND

ND

Cae

rset

al.

(2002)

ND

ND

Utt

ing

(1993)

ND

ND

Langdon

&W

aldock

(1981)

Nan

noch

loris

atom

us

6.4

54.5

9.1

112.9

44.1

3%

FAM

E

0%

FAM

ELa

ing

etal

.(1

990)

0%

TFA

0.1

%TF

AM

oure

nte

etal

.(1

990)

*V

alues

calc

ula

ted

from

refe

rence

dat

aif

not

direc

tly

report

edin

resu

lts.

�Fro

mLe

vens

&So

rgel

oos

(eds)

(1996).

�Ass

um

esca

rbohyd

rate

=4

calm

g)

1,

lipid

=9.4

calm

g)

1,

pro

tein

=4.5

calm

g)

1.

ND

,N

one

det

ecte

d;

FAM

E,fa

tty

acid

met

hyl

este

rs;

TFA

,to

talfa

tty

acid

s.



Results and discussion

Live algae

Algal species

A relatively short gut-passage time allows bivalve larvae to

assimilate only the easily mobilized cytoplasmic fraction

of algal cells (Reinfelder & Fisher 1994). To facilitate mobi-

lization, factors that must be considered when selecting

a particular algal species are form, motility (Martınez-

Fernandez et al. 2004), size (Baldwin 1995; Baldwin &

Newell 1995), toxicity (Laabir et al. 2007) and the ability

of the larvae to trap, ingest, digest and assimilate the

alga (Lora-Vilchis & Maeda-Martinez 1997). Tables 2–5

summarize the results of the growth rates of larvae fed

various algal species from published studies on O. edulis,

C. gigas, V. philippinarum and P. maximus, respectively.

Shell growth rates in these tables were standardized to lm

day)1 and the tables display both high-growth and low-

growth conditions for each of the species.

Growth rates of larval O. edulis exceeding 9 lm day)1

were achieved using monospecific diets of Isochrysis

galbana Parke, Phaeodactylum tricornutum Bohlin and

Pavlova lutheri (Droop) J.C. Green (Ferreiro et al. 1990)

(Table 2). The highest reported growth rates were with

bispecific diets of I. galbana and P. lutheri (9.7 lm day)1

(Jonsson et al. 1999)) and I. galbana and Tetraselmis

suecica (Kylin) Butcher (9.8–12.6 lm day)1 (Helm et al.

1973; Holland & Spencer 1973)). The lowest growth rates

were reported for Skeletonema costatum (Greville) Cleve

(0.4 lm day)1), a species which larvae of O. edulis are

unable to ingest, most likely because of the presence of

long chains and spines (Ferreiro et al. 1990). Survival

rates of larval O. edulis were high (>90%) with I. galbana,

P. tricornutum and Chaetoceros calcitrans f. pumilus Tak-

ano, but highly variable with T. suecica, P. lutheri and

Rhodomonas sp. (Table 2). According to Jonsson et al.

(1999), I. galbana is particularly important to O. edulis

because only larvae fed this algal species as a dietary com-

ponent were able to metamorphose within the 12 days of

their experiment.

Larvae of C. gigas tend to have the best growth rates

(>4.5 lm day)1) when fed a bispecific feed combination

with C. calcitrans as one of the components (Table 3). In

contrast with the results in Table 3, Brown and Robert

(2002) reported a result in which live I. galbana (Tahitian

strain, T-iso) and C. calcitrans produced poor growth of

larval C. gigas (2.3 lm day)1). Their study, however, used

a non-f. pumulim strain of C. calcitrans that was more

than double the mass of the f. pumulim strain (34.7

pg cell)1 compared with 13.8 pg cell)1, respectively).

Larval C. gigas had poorer ingestion rates on this larger

strain (15%) than on the smaller one (70–100%) (Brown

& Robert 2002). Table 3 shows that diets including algae

from the genus Pavlova typically produce poor results,

particularly when fed monospecifically, although bispecific

diets that include P. lutheri with I. galbana and ⁄ or C. cal-

citrans can produce growth rates over 5 lm day)1 (Rico-

Villa et al. 2006). Live P. lutheri fed monospecifically

Table 2 Culture conditions of larval Ostrea edulis and associated growth and survival rates

Algal species Temperature

(�C)

Stocking

density

(larvae mL)1)

Duration

of the

study (days)

Survival

(%)

Average

growth rate

(lm day)1)

Reference

Skeletonema costatum – 1.5–2 11 79–81 0.4 Ferreiro et al. (1990)

Isochrysis galbana and Chaetoceros calcitrans 22–24 1 12 – 2.8 Jonsson et al. (1999)

I. aff. galbana (T-iso) 25 18 0–18 3.9 Gallager et al. (1986)

I. galbana and Pavlova lutheri and C. calcitrans 20–22 1 12 – 5.4 Jonsson et al. (1999)

Rhodomonas sp. – 1.5–2 13 0–95 6.2 Ferreiro et al. (1990)

P. lutheri – 1.5–2 13 5–96 6.3 Ferreiro et al. (1990)

P. lutheri 20–22 1 12 – 6.4 Jonsson et al. (1999)

I. galbana and Tetraselmis suecica 16 17 – 7.6 Labarta et al. (1999)

C. calcitrans – 1.5–2 13 96–98 7.8 Ferreiro et al. (1990)

I. galbana 20–22 1 12 – 8.5 Jonsson et al. (1999)

T. suecica – 1.5–2 13 0–100 8.7 Ferreiro et al. (1990)

P. lutheri – 1.5–2 11 91–96.5 9.1 Ferreiro et al. (1990)

I. galbana – 1.5–2 11 92.5–93.5 9.2 Ferreiro et al. (1990)

Phaeodactylum tricornutum – 1.5–2 11 91–96 9.2 Ferreiro et al. (1990)

I. galbana and P. lutheri 20–22 1 12 – 9.7 Jonsson et al. (1999)

I. galbana and T. suecica 23.5–24.5 0.6 4 – 9.8 Helm et al. (1973)

I. galbana and T. suecica – 2.7 12 – 9.8 Holland & Spencer (1973)

I. galbana and T. suecica 23.5–24.5 0.6 4 – 12.6 Helm et al. (1973)

Growth rate results have been standardized to daily growth in shell length (lm day)1) and sorted from low to high. –, Not reported.

R. Marshall et al.


produced low growth rates of approximately 1.5

lm day)1 (Ponis et al. 2003, 2008; Rico-Villa et al. 2006)

and even lower rates when the alga was preserved (Ponis

et al. 2003). Bispecific diets of C. calcitrans in combina-

tion with Emiliania huxleyi (Lohmann) W.W. Hay &

H.P. Mohler, Imantonia rotunda N. Reynolds (Ponis et al.

2006c), Pavlova (= Rebecca) salina (N. Carter) J.C. Green,

Pavlova pinguis J.C. Green, Pavlova sp. AC 538 (Algo-

bank-Caen code) or Pavlova sp. 251 (Algobank-Caen

code) (Ponis et al. 2006b) were all poor, with growth

rates below 3 lm day)1. In all of the studies that reported

survival rates of 88% or higher, all diets contained C. cal-

citrans monospecifically or in combination with other

algal species (Table 3). Those studies that reported low

survival rates (<50%) used diets containing mostly

Pavlova species (Ponis et al. 2006c; Rico-Villa et al. 2006).

Larvae of V. philippinarum had the highest growth rates

(>7 lm day)1) when fed a monospecific diet of Isochrysis

spp. (Yan et al. 2006), a mixture of I. galbana (T-iso)

with C. calcitrans (Laing et al. 1990; Laing & Utting 1994)

or a monospecific diet of C. calcitrans (Utting & Doyou

1992) (Table 4). Low growth rates were associated with

Nannochloris atomus Butcher, T. suecica (Laing et al.

1990) and Chlorella spp. (Yan et al. 2006). Nannochloris

sp. was also found to be a poor feed for larvae of the

hard clam (Mercenaria mercenaria Linnaeus), the Ameri-

can oyster (Crassostrea virginica Gmelin) and the angel-

wing clam (Cyrtopleura costata Linnaeus) (Tan Tiu et al.

1989).

Good growth rates of 6 lm day)1 or higher for larvae

of P. maximus were achieved mostly with mixed-species

diets (Table 5). Combinations of P. lutheri, I. galbana

(T-iso) and C. calcitrans (Delaunay et al. 1993); P. lutheri,

I. galbana (T-iso) and S. costatum (Robert et al. 1996);

monospecific C. calcitrans (Delaunay et al. 1993); and

C. calcitrans in combination with Pavlova sp. AC 250

(Ponis et al. 2006b) all produced high growth rates.

Monospecific diets of P. lutheri or T-iso or a mixed diet

of C. calcitrans with T-iso or Pavlova spp. typically yielded

medium growth results of 4–5 lm day)1 (Table 5).

Dunaliella tertiolecta Butcher appears to be a poor algal

species choice for larval P. maximus, producing a growth

rate of only 1.6 lm day)1 (Delaunay et al. 1993).

In general, I. galbana and C. calcitrans are acceptable

algal feeds for larvae of the bivalve species examined

above. This is not surprising given that they are recom-

mended as the principal algal diets for bivalve larvae in

several publications (e.g. Utting & Spencer 1991; Utting

& Doyou 1992). An interesting finding is the apparent

poor suitability of Pavlova species for larvae of C. gigas,

Table 3 Culture conditions of larval Crassostrea gigas and associated growth and survival rates


(�C)

Stocking

density

(larvae mL)1)

Duration

of the

study (days)

Survival

(%)

Average

growth rate

(lm day)1)

Reference

Unfed 25 5 14 33.67 0.2 Rico-Villa et al. (2006)

Chaetoceros calcitrans f. pumilum and Pavlova pinguis 24 5 14 8.3 0.3 Ponis et al. (2006b)

C. calcitrans f. pumilum and Rebecca salina 24 5 14 20.1 0.4 Ponis et al. (2006b)

Pavlova lutheri (preserved at 4�C) 24 5 12 19 0.6 Ponis et al. (2003)

T-iso 24–26 5 16 22 0.9 Brown & Robert (2002)

Tetraselmis suecica 24 5 11–12 – 1.5 Robert et al. (2001)

P. lutheri 24 5 12 72.5 1.5 Ponis et al. (2003)

P. lutheri 25 5 14 29.23 1.7 Rico-Villa et al. (2006)

C. calcitrans f. pumilum and Pavlova sp. AC 538 24 5 14 42 1.8 Ponis et al. (2006b)

C. calcitrans f. pumilum and Imantonia rotunda 24 5 14 – 2.0 Ponis et al. (2006c)

C. calcitrans f. pumilum and Emiliania huxleyi 24 5 14 – 2.5 Ponis et al. (2006c)

C. calcitrans f. pumilum and Pavlova sp. AC 251 24 5 14 57.7 2.8 Ponis et al. (2006c)

C. calcitrans f. pumilum 24 5 14 – 3.0 Ponis et al. (2006c)

C. calcitrans f. pumilum and Pavlova sp. AC 250 24 5 14 57.9 4.5 Ponis et al. (2006c)

P. lutheri and T-iso and C. calcitrans 24 5 11–12 – 4.5 Robert et al. (2001)

C. calcitrans f. pumilum 24 5 14 89.1 4.8 Ponis et al. (2006b)

C. calcitrans f. pumilum and Pseudoisochrysis paradoxa 24 5 14 – 5.0 Ponis et al. (2006c)

C. calcitrans f. pumilum and Diacronema vlkianum 24 5 14 – 5.0 Ponis et al. (2006c)

C. calcitrans and T. suecica 25 8–8.6 4 – 5.0 Utting (1986)

C. calcitrans f. pumilum and T-iso 24 5 14 – 5.5 Ponis et al. (2006b)

C. calcitrans and T. suecica 25 8–8.6 4 – 5.6 Utting (1986)

P. lutheri and C. calcitrans f. pumilum 24 5 12 88 5.6 Ponis et al. (2003)




Table 5 Culture conditions of larval Pecten maximus and associated growth and survival rates


(�C)

Stocking

density

(larvae mL)1)

Duration

of the

study (days)

Survival

(%)

Average

growth rate

(lm day)1)

Reference

Unfed 18 7.5 22 69.1 0.9 Delaunay et al. (1993)

Dunaliella tertiolecta 18 7.5 22 86.4 1.6 Delaunay et al. (1993)

Unfed 18 5.0 14 90.6 3.3 Ponis et al. (2006b)

Chaetoceros calcitrans f. pumilum

and Pavlova pinguis

18 5.0 14 92.3 3.3 Ponis et al. (2006b)

C. calcitrans f. pumilum and Pavlova sp. AC 538 18 5.0 14 92.2 3.8 Ponis et al. (2006b)

C. calcitrans f. pumilum and Rebecca salina 18 5.0 14 91.9 4.2 Ponis et al. (2006b)

T-iso 18 7.5 22 72.9 4.4 Delaunay et al. (1993)

Pavlova lutheri 18 7.5 22 74.3 4.5 Delaunay et al. (1993)


C. calcitrans f. pumilum and T-iso 18 5.0 14 88.6 5.0 Ponis et al. (2006b)


P. lutheri, T-iso and C. calcitrans 18 7.5 22 78.4 5.8 Delaunay et al. (1993)

Isochrysis galbana, P. lutheri

and Skeletonema costatum

18 22 0 6.5 Robert et al. (1996)

C. calcitrans 18 7.5 22 80.2 6.5 Delaunay et al. (1993)

I. galbana, P. lutheri and S. costatum 18 – 23 75 6.8 Robert et al. (1996)


I. galbana (T-iso) P. lutheri and C. calcitrans 17.3–18.3 3.4 22 52 – Andersen et al. (2000)

I. galbana (T-iso) P. lutheri and C. calcitrans 17.3–18.3 – 22 5 – Andersen et al. (2000)

I. galbana (T-iso) P. lutheri and C. calcitrans 17.3–18.3 – 22 25–58 – Andersen et al. (2000)

I. galbana (T-iso) P. lutheri and C. calcitrans 17.3–18.3 – 22 20–45 – Andersen et al. (2000)

I. galbana, P. lutheri, C. calcitrans, Chaetoceros

muelleri, S. costatum and T. suecica

18 5.4–23 Variable 23 – Torkildsen & Magnesen

(2004)


Table 4 Culture conditions of larval Venerupis philippinarum and associated growth and survival rates


(�C)

Stocking

density

(larvae mL)1)

Duration of

the study

(days)

Survival

(%)

Average

growth rate

(lm day)1)

Reference

Nannochloris atomus 25 10 – – 1.19 Laing et al. (1990)

Isochrysis aff galbana (T-iso) and

Chaetoceros calcitrans

15 12–16 – – 4 Laing & Utting

(1994)

Nannochloris sp. (dried) 25 10 – – 4.16 Laing et al. (1990)

Chlorella spp. 17.2–22.4 5, 10, 15 and 20 14 62–73 after 10 days 5 Yan et al. (2006)

Chlorella spp. 17.2–22.4 5–10 8 5.2 Yan et al. (2006)

Tetraselmis suecica (dried) 25 10 – – 5.34 Laing et al. (1990)

T. suecica 25 10 – – 5.49 Laing et al. (1990)

Isochrysis spp. and ⁄ or Chlorella spp. 17.2–22.4 15 or 20 14 62–73 after 10 days 6 Yan et al. (2006)

Isochrysis spp. and ⁄ or Chlorella spp. 17.2–22.4 15 14 62–73 after 10 days 6 Yan et al. (2006)

Isochrysis spp. and ⁄ or Chlorella spp. 17.2–22.4 20 14 62–73 after 10 days 6 Yan et al. (2006)

Isochrysis spp. and ⁄ or Chlorella spp. 17.2–22.4 10 14 62–73 after 10 days 6.4 Yan et al. (2006)

Isochrysis spp. and ⁄ or Chlorella spp. 17.2–22.4 5 or 10 14 62–73 after 10 days 6.7 Yan et al. (2006)

Isochrysis spp. and ⁄ or Chlorella spp. 17.2–22.4 5 14 62–73 after 10 days 6.9 Yan et al. (2006)

Isochrysis spp. 17.2–22.4 5, 10, 15 and 20 14 62–73 after 10 days 7 Yan et al. (2006)

I. galbana and C. calcitrans 25 10 – 8.46 Laing et al. (1990)

I. aff galbana (T-iso) and C. calcitrans 25 12–16 – 9.2 Laing & Utting

(1994)

C. calcitrans 25 11 6 36.8–54 13.3–13.7 Utting & Doyou

(1992)


R. Marshall et al.


particularly when the alga is fed monospecifically. Pavlova

lutheri, although recommended as a feed for Crassostrea

species instead of T-iso (owing to a more favourable poly-

unsaturated fatty acid (PUFA) content) (Helm et al.

2004), appears to be incompatible with larval C. gigas.

Larvae of C. gigas ingest P. lutheri (Ponis et al. 2003;

Rico-Villa et al. 2006), and the similar species P. pinguis

and P. salina (Ponis et al. 2006b), poorly. Ingestion,

growth and survival rates of larval C. gigas fed Pavlova

species can be increased by combining Pavlova with other

algae (Ponis et al. 2003; Rico-Villa et al. 2006). However,

increasing ingestion rates of P. lutheri do not ensure

success, as larvae of C. gigas fed with a combination of

P. lutheri and T-iso grew well, but failed to produce

pediveligers (Rico-Villa et al. 2006). The poor perfor-

mance with Pavlova species is not, however, the result of

toxicity, as determined by feeding larvae of C. gigas with

supernatant from centrifuged algal cultures (Ponis et al.

2003, 2006b).

Poor results have also been reported for other bivalve

species when fed Pavlova spp. Larvae of the Asiatic hard

clam (Meretrix meretrix Linnaeus) had reduced growth

rates when fed Pavlova viridis C.K. Tseng, J. Chen &

X. Zhang compared with I. galbana (Tang et al. 2006) and

larvae of O. edulis fed P. lutheri accumulated less lipid

compared with individuals fed I. galbana, P. tricornutum

or S. costatum monospecifically (Ferreiro et al. 1990).

Pernet et al. (2003) showed that larvae of the sea scallop

(Placopecten magellanicus Gmelin) fed P. lutheri had poor

survival, but larval performance was markedly improved

in later studies by replacing P. lutheri with Pavlova sp.

(strain CCMP 459) (Pernet et al. 2005). Pavlova lutheri is

not a poor feed for all bivalve species, however, producing

good growth rates when fed to larval Sydney rock oysters

(Saccostrea commercialis Iredale and Roughley) (Nell &

O’Connor 1991) and larval P. maximus (Delaunay et al.

1993; Ponis et al. 2003, 2006b).

Despite the predominant use of a select few species of

algae for hatchery production, there is evidence that wild

species of algae native to the region of the cultured

bivalve species may yield better growth and survival than

standard laboratory species. Gouda et al. (2006) isolated

and reared the wild plankton Prymnesium sp., Navicu-

la pelliculosa (Brebisson ex Kutzing) Hilse and Chaetocer-

os septentrionalis Ostrup, and subsequently fed them to

larval P. magellanicus. Compared with T-iso, P. lutheri

and C. calcitrans, they produced superior survival (40%

for wild algae compared with 30% for laboratory algae

after 35 days) (Gouda et al. 2006). There has been some

recent research investigating the potential of new algal

species for larval rearing (Martınez-Fernandez et al. 2006;

Pernet et al. 2005; Ponis et al. 2006b,c), but the process

of isolating and testing new species of algae is very labour

intensive and hatcheries are unlikely to adopt new algal

species without well-established culture methods and

proven long-term success.

Algal size and digestibility

Another important factor in larval bivalve nutrition is the

size of the algal cells (Table 6). The maximum cell size

that can be ingested is related to larval body size (Lora-

Vilchis & Maeda-Martinez 1997). Clearance rates for

bivalve veligers are typically highest with particle sizes

between 4.7 and 6.3 lm (Sommer et al. 2000), although

they are capable of ingesting cells well outside this range

(up to 16 lm for <150 lm larvae and up to 30 lm for

>200 lm larvae) (Baldwin & Newell 1995). This means

that as larvae grow, the appropriate-sized algae must be

provided in relation to the stage of development to

ensure proper ingestion.

Table 6 Larval size of various bivalve species and the maximum ingestible algal size for the given shell length

Larval size,

shell length

(lm)

Algal size

(lm)

Algal species Bivalve species Reference

80 1.5–2 Nannochloris oculata Argopecten ventricosus-circularis Lora-Vilchis & Maeda-Martinez (1997)

90 3–5 Isochrysis galbana Argopecten ventricosus-circularis Lora-Vilchis & Maeda-Martinez (1997)

Monochrysis lutheri

Pavlova lutheri

Phaeodactylum tricornutum

Thalassiosira pseudonana (3H)

100 4–6 Chaetoceros calcitrans Argopecten ventricosus-circularis Lora-Vilchis & Maeda-Martinez (1997)

Chaetoceros muelleri

120 4–10 (with 35 lm

chains plus spines)

Chaetoceros septentrionalis Placopecten magellanicus Gouda et al. (2006)

140 12–15 Tetraselmis suecica Venerupis philippinarum Utting & Doyou (1992)

Thalassiosira pseudonana (S78)



Several examples of the ability of larvae to ingest cells

of increasing size with growth have been described. Lar-

vae of C. gigas, for example, grow poorly on a monospe-

cific diet of T. suecica (1.2–1.7 lm day)1) until reaching

140 lm, after which growth is much improved (6.4–

8.0 lm day)1) (Robert et al. 2001). This supports the

assertion that T. suecica should not be used until larvae

reach 120 lm in shell length (Utting & Spencer 1991;

Utting & Doyou 1992). Similar results were seen with

C. septentrionalis fed to larval P. magellanicus, where

growth rates doubled once the larvae exceeded 120 lm

(Gouda et al. 2006). Veligers of the catarina scallop

(Argopecten ventricosus-circularis Sowerby) can ingest cells

of Nannochloropsis oculata (Droop) D.J. Hibberd (1.5–

2.0 lm) within the first day of becoming a D-larva, but

they require an extra day of growth when fed I. galbana,

P. lutheri, P. tricornutum (3–4 lm) or Thalassiosira

pseudonana Hasle & Heimdal (4–5 lm) (Lora-Vilchis

& Maeda-Martinez 1997). Larvae of P. maximus and

C. virginica can ingest P. lutheri (3–4 lm), however,

within the first days after embryogenesis (Babinchak &

Ukeles 1979; Le Pennec & Rangel-Davalos 1985). Shell

heights of approximately 95 lm are required for larvae of

A. ventricosus-circularis to be able to ingest the spiny,

chain-forming diatoms C. calcitrans (4–5 lm) and Chae-

toceros muelleri Lemmermann (5–6 lm), whereas this

species was not able to ingest D. tertiolecta (5–6 lm),

T. suecica (8–9 lm) or T. pseudonana (10–11 lm) within

the first 7 days of life (Lora-Vilchis & Maeda-Martinez

1997).

In addition to size characteristics, Lora-Vilchis and

Maeda-Martinez (1997) suggested that algal motility

might also be a factor contributing to a larva’s ability to

capture prey items. They described this with respect to

D. tertiolecta fed to larvae of A. ventricosus-circularis;

ingestion of the algae was poor and the authors suggested

that the rapid movement of the prey algae may have

made it difficult for the larvae to capture the cells. This

was not quantified, but there is some evidence that poor

ingestion of Dunaliella species may be common because it

was also reported for larvae of P. maximus (Le Pennec &

Rangel-Davalos 1985) and the winged pearl oyster (Pteria

sterna Gould) (Martınez-Fernandez et al. 2004). Research

on the motility of algae is under-represented in the litera-

ture, but as it may have a direct relationship to its value

as a food item it may be worth examining as a field of

research.

The digestibility of algae is also an important factor in

larval nutrition and is related to the cell-wall structure

and morphology of the algae. Easily digested algae include

naked flagellates such as I. galbana and P. lutheri

(Le Pennec & Rangel-Davalos 1985; Lora-Vilchis & Maeda-

Martinez 1997; Martınez-Fernandez et al. 2004). Diatoms

with large spines, such as T. pseudonana and C. calcitrans,

are also generally readily digested, but those that form

large chains, like P. tricornutum, are not (Lora-Vilchis &

Maeda-Martinez 1997). Nannochloropsis species are also

not well digested, despite being easily ingested, possibly

because of the fibrous glycoprotein cell wall (Lora-Vilchis

& Maeda-Martinez 1997; Martınez-Fernandez et al. 2004).

The thick-cell-walled Chlorella autotrophica Shihira &

Krauss is also poorly digested (Babinchak & Ukeles 1979)

and Chlorella species in general are considered to be

difficult to digest (Helm et al. 2004).

Biochemically modified algae

Culturing algae under varying conditions can modify

their biochemical composition (Utting 1985; Thompson

et al. 1990, 1992; Ponis et al. 2006a). In addition, the

fatty acid content of algae can be directly manipulated

by adding fatty acid supplements to algal culture water

(von Elert 2002; Seguineau et al. 2005). Manipulation of

algae may be a relatively simple way to alter feed com-

positions without culturing several species, allowing the

culturist to focus on algal species that are known to have

high ingestion and digestion rates. Factors that can affect

cellular composition include light intensity and tempera-

ture (which can influence fatty acid levels) (Thompson

et al. 1990, 1992; Thompson & Harrison 1992), nitrogen

levels in the culture media (which can alter protein con-

centration) (Gallager & Mann 1981; Enright et al. 1986;

Utting 1986) and silica levels in diatom culture media

(which can affect TAG levels) (Enright et al. 1986). Sur-

vival does not seem to be influenced by algal modifica-

tion, but growth can be affected, as shown in larval blue

mussels (Mytilus edulis Linnaeus) (Leonardos & Lucas

2000).

Thalassiosira pseudonana exposed to high light levels

of 110 lmol photons m)2 s)1 had higher DHA levels of

3.1% of total fatty acids (TFA) compared with 2.5% of

TFA at 13 lmol photons m)2 s)1 (Thompson & Harri-

son 1992). Altering the light intensity may decrease the

relative content of EPA, however, as seen in Chaetoceros

simplex Ostenfeld (Thompson et al. 1990) and T. pseudo-

nana (Thompson & Harrison 1992). However, this

reduction in EPA may not be a concern as Thompson

and Harrison (1992) found that feeding T. pseudonana

grown in a high light intensity to larval C. gigas

improved both growth and survival rates compared with

larvae fed T. pseudonana grown in a low light intensity.

Temperature can also influence the relative levels of fatty

acids; monounsaturated fatty acids are a higher percent-

age of the TFA at high temperatures (25�C) compared

with low temperatures (10�C), which produce higher

levels of polyunsaturated fatty acids (Thompson et al.

1992).

R. Marshall et al.


Modified lipid levels in diatoms have been achieved by

limiting silica in the culture media. Limitation of silica

reduces growth rates of the algae, but TAG accumulation

is enhanced because the energy used for silica uptake is

diverted to lipid production (Lombardi & Wangersky

1991). Enright et al. (1986) used f ⁄ 2 media without sili-

cate, which doubled the lipid content per cell of Chaeto-

ceros gracilis F. Schutt, and improved growth rates of

juvenile O. edulis in an otherwise nutrient-limited envi-

ronment. Triacylglycerol-enriched C. muelleri increased

the shell length of larvae of P. magellanicus when fed in

combination with I. galbana compared with a diet of

I. galbana and C. muelleri cultured under normal condi-

tions (Pernet et al. 2003).

One aspect that has not been investigated is the effect

of silica limitation on algal digestibility. There is a possi-

bility that, under silica deprivation, diatom tests are less

developed, allowing for easier ingestion owing to short-

ened or more fragile spines and ⁄ or easier digestion

because of a thinner test wall. Research in this area may

be beneficial. If digestibility can be improved with

diatoms, by providing less silica for test development,

then species such as P. tricornutum and S. costatum

(which are known to have favourable nutritional compo-

sition, but poor ingestibility and digestibility) may be

made more suitable for larval culture.

Protein levels in algae are dictated by the levels of

nitrogen that they are exposed to in culture. In a nitro-

gen-poor environment (0.613 mg atoms L)1) compared

with a normal nitrogen environment (9.8 mg atoms L)1),

I. galbana, C. calcitrans and T. suecica all had reduced

protein levels (Utting 1985), as did T. pseudonana under

similar conditions (Gallager & Mann 1981). High light

levels have also been shown to reduce the protein concen-

tration in T. pseudonana (Gallager & Mann 1981;

Thompson et al. 1990). High-protein T-iso combined

with high-protein Chaetoceros neogracile S.L. VanLanding-

ham (= C. gracilis F. Schutt) provided better growth and

survival than normal T-iso and C. neogracile (5.7

lm day)1 and 4.7 lm day)1, respectively) in larval

Chilean scallops (Argopecten purpuratus Lamarck) (Uriarte

et al. 2004).

It is important to note that if techniques are used to

modify the content of one biochemical component, the

levels of other components may be affected. Utting

(1985), for example, showed that reducing the levels of

protein in algae increased the levels of carbohydrate and

lipid. For this reason, modified algae should probably be

used in conjunction with unmodified algae to ensure

nutritional balance in the diet.

Direct enrichment of algae with specific fatty acids

could be a very simple way to improve algal nutritional

quality. Studies have shown that EPA, DHA and AA

levels in Scenedesmus obliquus MEYEN can be increased

from non-detectable amounts to 10–20% of the mea-

sured fatty acids by directly adding fatty acids to the

culture (von Elert 2002). It can take less than 4 h for

algae to become enriched with specific fatty acids as

demonstrated with Stephanodiscus hantzschii GRUNOW

enriched with a-linolenic acid (von Elert 2002) and T-iso

enriched with AA (Seguineau et al. 2005). Despite the

ease with which this process is accomplished, the results

of Seguineau et al. (2005) suggested that there might be

biological changes to enriched algae that make it less

suitable as food. In their study, C. gigas spat fed enriched

algae (T-iso) had reduced size and condition compared

with spat fed untreated T-iso. Whether or not this

negative applies to larval bivalves is not known. Nor is it

known if the technique is more suitable to other algae

species or specific fatty acids. Research in this area

should be conducted.

Other feeds

Preserved and concentrated algae

The culture of live algae at a hatchery site is a costly and

risky method of feeding larvae. One potential replacement

for this type of feeding system is concentrated algal paste.

This product is formed by concentrating algal cells

(through centrifugation, filtration or flocculation) from

mass cultures and preserving the resultant paste (through

refrigeration, freezing or drying) (Grima et al. 1994;

Brown & McCausland 2000; Ponis et al. 2008). The issue

is whether or not this type of feed is adequate for larval

culture. Table 7 shows results from several experiments

using preserved live feeds. The lowest growth rates (0.6

lm day)1) and survival rates (19%) are attributable to pre-

served P. lutheri fed to larval C. gigas (Ponis et al. 2003).

For C. gigas, live feeds tend to provide better growth and

survival, but adequate growth rates of 4.6 lm day)1 were

found with live T-iso and a concentrate of C. calcitrans

(forma pumulin) (Brown & Robert 2002). In this case it

appears that the forma Pumulin strain of C. calcitrans was

important to success because the other C. calcitrans strain

used produced poor growth rates of 2.0 lm day)1. Tetra-

selmis appears to be much better suited as a preserved feed

because dried and live T. suecica produced similar growth

rates of 5.3 lm day)1 and 5.5 lm day)1, respectively,

when fed to larval V. philippinarum (Laing et al. 1990)

and cold-stored Tetraselmis produced very similar growth

rates to fresh Tetraselmis when fed in combination with

live feed to larval C. gigas (Robert et al. 2001).

A potential advantage of T. suecica as a preserved feed

is that it may be grown heterotrophically. Heterotrophi-

cally grown algae use organic carbon instead of light as

an energy source (Coutteau & Sorgeloos 1993) and are



grown in fermenters (Laing et al. 1990). Most algae

require light to grow, so this technique is limited to very

few species (most commonly T. suecica and Schizochytri-

um sp.) (Coutteau & Sorgeloos 1993). This technique

costs approximately 30% of what it costs to produce algae

autotrophically and results in lower variation in the

chemical composition (Utting 1993), and development of

the culture methods could improve the nutritional value

(Jones et al. 1993).

Formulated feeds

The use of non-algal and formulated feeds, including yeast

(Epifanio 1979; Coutteau et al. 1994), flour (Albentosa

et al. 1999), cheese-whey (Enes & Borges 2003), gelatine

acacia spray beads (Novoa et al. 2002) and emulsions and

liposomes (Coutteau & Sorgeloos 1993; Caers et al. 1999),

has been investigated for bivalves for many years. Nearly

all published studies, however, have looked at the use of

these diets with juvenile bivalves. There have been some

larval studies looking at the use of lipid microcapsules

(Chu et al. 1982, 1987) and lipid emulsion spheres

(Hendriks et al. 2003). Despite this lack of research we

have examined the available literature to try to shed some

light on this subject.

The use of PUFA spheres, as a single feed source, does

not appear to be particularly beneficial at the larval stage.

Table 7 Growth and survival rates of bivalve larvae fed various forms of preserved and artificial feeds (compared with live feeds)

Species Feed type Survival

(% surviving

at the end of the

trial unless

otherwise noted)

Average

growth

rate

(lm day)1)

Feed rate Reference

Preserved feeds

Crassostrea gigas Pavlova lutheri (concentrate preserved

at 4�C)

19 0.6 Three times

per week

Ponis et al.

(2003)

P. lutheri (fresh concentrate) 67–78 1.5

P. lutheri and Chaetoceros calcitrans

forma pumilum (live)

87–89 5.6

C. calcitrans forma

pumilum (live)

86 6.1

C. gigas T-iso and Skeletonema spp. (concentrate) 51 1.8 Daily Brown &

Robert (2002)T-iso and C. calcitrans (concentrate) 61 2.0

T-iso and C. calcitrans (live) 73 2.3

T-iso and Skeletonema spp. (concentrate) 73 3.1

T-iso and C. calcitrans (f.p.) (concentrate) 52 4.6

C. gigas PTC and Tetraselmis sp. (concentrate

4-day-old preserved)

– 5.4 Every

2 days

Robert et al.

(2001)

PTC and Tetraselmis sp. (concentrate fresh) – 5.7

C. calcitrans and Tetraselmis suecica (live) 92 7.2

Venerupis

philippinarum

Nannochloris atomus (live) – 1.2 Daily Laing et al.

(1990)Nannochloris sp. (dried) – 4.2

T. suecica (dried) – 5.3

T. suecica (live) – 5.5

C. calcitrans and T-iso (live) – 8.5

Artificial feeds

Saccostrea

commercialis

Unfed 89.8 2.2 Unfed Southgate

et al. (1992)Microcapsules (chicken egg ovalbumin,

fish roe, cod liver oil)

99.1 6.6 Daily

Microcapsules (chicken egg ovalbumin,

fish roe, cod liver oil) and yeast extract

89.8 6.7 Daily

Isochrysis aff galbana (T-iso) and C. calcitrans

and P. lutheri (live)

95.9 8.2 Daily

Macoma balthica Lipid emulsion PUFA spheres 4.73% mortality day)1 0.6 Hendriks et al.

(2003)Unfed 4.10% mortality day)1 1.84

Instant algae (preserved Isochrysis spp.)

and lipid emulsion PUFA spheres

4.23% mortality day)1 3.1

Instant algae (preserved Isochrysis spp.) 3.98% mortality day)1 5.3

PTC, live Pavlova lutheri, Isochrysis galbana and Chaetoceros calcitrans; PUFA, polyunsaturated fatty acid. –, Not reported.

R. Marshall et al.


The data summarized in Table 7 show the results of sev-

eral studies using formulated feeds in comparison to con-

trols. Larvae of Macoma balthica Linnaeus fed solely with

lipid enrichment emulsions (DHA ⁄ EPA) showed very

poor growth (0.6 lm day)1), no better than unfed con-

trols (Hendriks et al. 2003). The same study found that

high larval growth rates could be achieved when lipid

spheres were used as a supplement to live feed. A promis-

ing enriched feed appears to be microcapsules containing

chicken ovalbumin, fish roe and cod liver oil; this feed

produced very favourable growth rates of 6.6 lm day)1 in

larval S. commercialis (Southgate et al. 1992). Southgate

et al. (1992) also found that the addition of yeast extract

did not significantly improve larval growth rates. This

formulation was most likely successful because it was

derived from extracts of natural food sources that have a

full complement of biochemical components. Simply tar-

geting particular nutritional components such as EFAs

does not supply complete nutrition, as shown by Hend-

riks et al. (2003) and, for this reason, lipid and EFA

microcapsules and ⁄ or emulsion spheres are suitable only

as supplements to live feed.

Another potential method for delivering formulated

supplements is by directly adding dissolved organic

matter to the culture water. Recent work by Seguineau

et al. (2005) suggests that the direct addition of fatty

acids to C. gigas spat culture tanks at the same time that

algae are added does not negatively affect the spat and

that they will assimilate the fatty acid. This result sup-

ports the work of Bunde and Fried (1978) who showed

that the 14C-labelled form of palmatic acid could be

extracted from seawater by adult C. virginica. Direct use

of dissolved supplements with larvae is largely unex-

plored, but there is evidence that it may be beneficial.

Crassostrea gigas larvae showed improved growth rates

when the dissolved organic fractions of an artificial feed

derived from egg albumin, rice starch, oyster lipid

extract, phosphorus, yeast RNA and salmon sperm DNA

were added to the culture water (Langdon 1983). The

study did not determine which components of the

fraction contributed to the improved growth. Molluscan

larvae have been shown to directly uptake sugars

(Welborn & Manahan (1990) for C. gigas) and amino

acids (Manahan (1983) for C. gigas and M. edulis) from

seawater. The uptake of fatty acids by bivalve larvae is

not well researched, but there are examples from other

invertebrates, such as the non-feeding larvae of the

sponge Tedania ignis (Porifera, Demospongiae), which

can transport palmitic acid across the epidermis (Jaeckle

1995). The simplicity of this technique for the delivery

of nutrient supplements is very appealing from a culture

perspective. Further research is warranted on the ability

of larvae to uptake specific nutrients, the rates of

uptake and the effects that they may have on growth

and survival.

Alternative live feeds (bacteria and picoplankton)

Standard algal species are grown almost exclusively as

feeds at most hatcheries, but there is some evidence that

certain species of bacteria (Douillet 1993; Douillet &

Landgon 1993) and picoplankton (Baldwin 1995) may be

beneficial to bivalve larvae, possibly by supplying

micronutrients such as vitamins (Seguineau et al. 1996).

Larvae of C. gigas and the Japanese pearl oyster (Pinct-

ada fucata martensii Dunker) are capable of ingesting

bacteria and picoplankton (Douillet 1993). Without any

supplemental feed, larvae of C. gigas were found to live

for prolonged periods (up to 33 days), while still main-

taining metabolic rates, presumably surviving on bacteria

(Moran & Manahan 2004). Many bacterial strains are det-

rimental to larvae, but the bacterial strain CA2 (isolated

from Netarts Bay in Oregon) consistently improved the

survival and growth of larvae of C. gigas by 22 and 21%,

respectively, when added with I. galbana at 10 000 or

100 000 cells mL)1 (Douillet & Landgon 1993). Similarly,

I. galbana supplemented with Synechococcus sp. bacteria

enhanced the growth of larval M. mercenaria, but larvae

performed poorly with a straight bacterial monoculture

(Gallager et al. 1994). An unidentified picoplankton

bloom enhanced the growth of larvae of P. maximus that

were being fed algae of low nutritional value (Ponis

et al. 2006b). Despite the apparent benefits, very little

research has been done on the effects of bacteria and

picoplankton as feed supplements for bivalve larvae. Each

target species of bivalve and bacteria or picoplankton

would have to be tested to confirm which were beneficial

and which would be economically viable from a commer-

cial hatchery perspective. This may be a fruitful avenue of

research.

Feed ration (proteins, carbohydrates, lipids and essential

fatty acids)

There have been a number of studies examining the use

of proteins, lipids and carbohydrates by larval bivalves.

Studies have shown in larvae of O. edulis (Holland &

Spencer 1973) and the rock scallop (Crassadoma gigantea

J. E. Gray) (Whyte et al. 1992) that, when starved, nearly

half of the energy used comes from lipid reserves, fol-

lowed by protein (30–40%) and carbohydrate (<10%).

Triacylglyceride reserves are of particular importance,

accounting for up to 80% of lipid loss in C. virginica,

O. edulis and M. mercenaria during starvation, and high

levels are correlated with high survival rates (Gallager

et al. 1986). It appears that protein has a more important

role in late larval stages and during metamorphosis, with



the highest amount of energy contributed by protein

occurring during settlement in O. edulis (Rodrıguez et al.

1990) and C. gigas (Bartlett 1979). Carbohydrate, in addi-

tion to protein, may also play a significant role during

larval metamorphosis as shown in C. gigas and C. virgi-

nica (Haws et al. 1993).

Despite the relationships between the biochemical

requirements of bivalve larvae and their growth and sur-

vival, little has been done in the way of determining what

levels need to be supplied to larvae on a daily basis to

provide optimum performance. Some preliminary work

was done by Utting (1986) on correlations between larval

0

2

4

6

8

10

12

14

0 7500 15 000 22 500 30 000 37 500 45 000

(c)

0

2

4

6

8

10

12

14

0 1000 2000 3000 4000 5000 6000

(d)

0

2

4

6

8

10

12

14

0 20 000 40 000 60 000 80 000 100 000 120 000

(b)

0

2

4

6

8

10

12

14

0 750 1500 2250 3000 3750 4500 5250

(e)

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0 2 4 6 8 10 12 14 16 18 20 22 24 26

(f)

0

2

4

6

8

10

12

14

0 40 000 80 000 120 000 160 000 200 000

(a)

Figure 2 Growth rates of larvae of Crassostrea gigas in relation to ingested levels of various feed components (level ingested larva)1 day)1).

(a) Protein, (b) lipid, (c) carbohydrate, (d) eicosapentanoic acid (EPA), (e) docosahexaanoic acid (DHA) and (f) energy. Data represent a compilation

of standardized results from Utting (1986), His et al. (1989), His & Seaman (1992), Robert et al. (2001), Brown & Robert (2002), Ponis et al.

(2003, 2006b,c), Rico-Villa et al. (2006) and Taris et al. (2006). The lines represent piecewise spline regression models.

R. Marshall et al.


growth rates and the levels of protein, carbohydrate, lipid

and total energy in larval diets, but relatively little has

been done since. To build on this relationship, growth

rate results from studies on larval C. gigas were compiled

and analysed. Figure 2 shows the correlations between lar-

val growth rates and the levels of various biochemical

components ingested (protein, lipid, carbohydrate, EPA,

DHA and energy) and Table 8 displays the strength of

these correlations as determined by linear regression. The

linear regressions of the growth rates (ln transformed)

with the various ingested biochemical compounds are all

highly significant with correlation coefficients (r) ranging

from 0.44 to 0.76 (Table 8). In general, these trends show

that there is an increase in growth rates with increasing

levels of the biochemical components ingested up to a

limit, after which growth rates drop. This may show a

point where overfeeding becomes a problem, with excess

food leading to waste accumulation, tank fouling and

poor larval performance (Utting & Spencer 1991). There

is the possibility that poor performance caused by over-

feeding may be avoided through algal modification or

feed supplements that provide increased levels of bio-

chemical components per algal cell, improving the nutri-

tional value while avoiding the introduction of excess

feed.

Table 9 shows the maximum effective dosages of each

biochemical component, as determined by piecewise lin-

ear spline modelling, and the algal cell equivalent for sev-

eral common algal species. The concentration of lipids

required for maximum growth rate of larval C. gigas,

based on the results of the analysis, is approximately

50 000 pg larva)1 day)1 (Table 9). This can be supplied

by a daily ration per larva of approximately 8500 cells of

T-iso, 28 000 cells of C. calcitrans forma pumilus, 3000

cells of T. suecica or 4100 cells of P. lutheri (see Tables 1

and 9). The ration for C. calcitrans is a high number of

cells, but given its relatively small mass (approximately

half that of T-iso), this may be a level that could

be ingested by C. gigas larvae and is in line with the

cell counts provided in several studies (Robert et al. 2001;

Brown & Robert 2002; Rico-Villa et al. 2006). As P. luthe-

ri stimulates low ingestion rates and poor larval success

in C. gigas, P. lutheri is not recommended for this species.

Triacylglycerol levels in larval food correlate positively

with growth in species other than C. gigas, as has been

shown in P. magellanicus (Pernet et al. 2003), and if

properly fed, the lipid levels of larvae will increase during

larval growth (Hendriks et al. 2003).

The lipid content of the diet is important to larval

development, but EFAs appear to be a limiting factor.

High lipid diets fed to larval A. purpuratus (Nevejan et al.

2003b) and P. maximus (Delaunay et al. 1993) both

resulted in poor growth when EPA and DHA were

absent. Similarly, Thompson and Harrison (1992) found

that increasing DHA levels in T. pseudonana from 1.1 to

3.1% of TFA improved the growth of larval C. gigas. The

dose–response analysis suggests that for larval C. gigas

2270 pg larva)1 day)1 EPA and 533 pg larva)1 day)1

DHA are adequate to attain maximum rates of growth

(Table 9). This means that the low-EPA species T-iso is

a poor candidate for monospecific feeding, requiring

approximately 90 000 cells larva)1 day)1 to reach suffi-

cient EPA levels. This is a daily cell ration that is much

higher than the highest reported value in the published

studies reviewed here, which was a combined total of

24 000 cells of P. lutheri and T-iso larva)1 day)1 (Rico-

Villa et al. 2006). In contrast, Chaetoceros calcitrans

requires only 19 700 cells larva)1 day)1 to supply the

needs for both EFAs (EPA and DHA), making it a more

suitable candidate for monospecific feeding. This level is

reflected in studies in which larvae of C. gigas were fed

monospecifically with C. calcitrans (f. pumilum). Ponis

Table 8 Linear equations correlating daily growth rate of larval Crassostrea gigas to the levels of various components of the feed�

Component Linear equations Observations

(n)

Correlation

coefficient (r)

Slope significance

(P-value)

Protein ln(g + 1) = ()2.1358) + (0.3535) · ln(p + 1) 59 0.76 <0.0001

Lipid ln(g + 1) = ()1.3038) + (0.3091) · ln(l + 1) 59 0.72 <0.0001

Carbohydrate ln(g + 1) = ()1.1377) + (0.2990) · ln(c + 1) 59 0.67 <0.0001

EPA ln(g + 1) = (0.4639) + (0.1927) · ln(e + 1) 59 0.59 <0.0001

DHA ln(g + 1) = (1.0331) + (0.1294) · ln(d + 1) 59 0.44 <0.0001

Energy ln(g + 1) = (0.7716) + (0.5094) · ln(en + 1) 59 0.76 0.0005

�Growth rate = average daily increase in shell length of larvae (lm day)1).

g, growth rate; p, pg protein larva)1 day)1; l, pg lipid larva)1 day)1; c, pg carbohydrate larva)1 day)1; e, pg eicosapentanoic acid (EPA) larva)1

day)1; d, pg docosahexanoic acid (DHA) larva)1 day)1; en, 10)4 calories larva)1 day)1.

Analysis based on a compilation of results from Utting (1986), His et al. (1989), His and Seaman (1992), Robert et al. (2001), Brown & Robert

(2002), Ponis et al. (2003, 2006a,b), Rico-Villa et al. (2006) and Taris et al. (2006).



et al. (2006c) reported low growth rates of 3 lm day)1

at a ration of 10 000 cells larva)1 day)1 with growth

rates nearly doubling (5.8 lm day)1) at a ration of

20 000 cells larva)1 day)1 (Ponis et al. 2006b) and very

high growth rates (10 lm day)1) at 24 000 cells larva)1

day)1 (Rico-Villa et al. 2006). Chaetoceros calcitrans is

also a successful monospecific feed for larval P. maximus

(Delaunay et al. 1993) and V. philippinarum (Utting

& Doyou 1992). Based on dose–response curves it

becomes clear why combination diets are superior

(Utting & Spencer 1991; Utting & Doyou 1992) at

meeting the dose requirements for both EPA and DHA.

Rico-Villa et al. (2006) showed that unbalanced ratios

of T-iso ⁄ C. calcitrans (95:5 and 5:95) led to unbalanced

EFA levels in larval C. gigas and poor larval settlement.

It is important to note that the omega-6 fatty acid

DPA, although not included in the piecewise regression

analysis, may be a potentially important fatty acid.

Placopecten magellanicus larval growth and survival were

both improved when fed Pavlova sp. (strain CCMP

459), a high DPA species, compared with the low DPA

species P. lutheri (Pernet et al. 2005). The authors sug-

gested that DPA was particularly important during early

veliger development when this fatty acid was accumu-

lated. The reason for the accumulation was unclear, but

the authors suggested that it might act as a homologue

of DHA. Docosapentanoic acid has also been identified

as an important nutrient in the post-larval stage of

P. magellanicus by (Milke et al. 2008).

Protein is a biochemical component that is rarely dis-

cussed in terms of larval bivalve growth, but a high pro-

tein diet has been linked to improved larval growth and

survival in both C. gigas and A. purpuratus (Utting

1986; Uriarte et al. 2004). The protein level necessary

for optimal larval C. gigas growth, estimated by a dose–

response analysis, is 69 200 pg larva)1 day)1, a level that

can be supplied by 10 200 cells larva)1 day)1 I. gal-

bana (T-iso) or 18 200 cells larva)1 day)1 C. calcitrans

(Table 9). Other larger-celled species, such as P. tricor-

nutum (3000 cells larva)1 day)1), S. costatum (5300 cells

larva)1 day)1), T. suecica (1300 cells larva)1 day)1) and

D. tertiolecta (3500 cells larva)1 day)1), require lower

cell counts to supply the required protein, but are only

appropriate for larger (>120 lm) larvae. The identified

level compares favourably with the level of 50 000–

100 000 pg protein larva)1 day)1 identified for larval

C. gigas (Utting 1986). Utting (1986) also suggested that

a protein ration of 250 000 pg larva)1 day)1 would

improve settlement rates.

The estimated overall energy requirement for larvae of

C. gigas is 9.75 · 10)4 calories larva)1 day)1 (Table 9), a

level easily supplied by most of the algal species listed.

Based on the analysis it does not appear that energyTab

le9

Max

imum

effe

ctiv

edose

–res

ponse

sof

various

bio

chem

ical

com

ponen

tsan

den

ergy

for

larv

alC

rass

ost

rea

gig

asan

das

soci

ated

rations

of

algae

that

will

mee

tth

em

axim

um

dose

–

resp

onse

s

Com

ponen

tC

orr

elat

ion

coef

fici

ent

(r)

Cla

ssH

apto

phyc

eae

Bac

illar

iophyc

eae

Pras

inophyc

eae

Chlo

rophyc

eae

Spec

ies

I.gal

ban

a(T

-iso

)P.

luth

eri

C.

calc

itra

ns

P.tr

icorn

utu

mS.

cost

atum

T.su

ecic

aD

.te

rtio

lect

a

Max

imum

effe

ctiv

edose

(pg

larv

a)

1day

)1)

(ener

gy

inca

lories

larv

a)1

day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)

1day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)

1day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)

1day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)1

day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)1

day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)

1day

)1)

(±95%

CI)

Max

imum

effe

ctiv

e

ration

(cel

ls

larv

a)

1day

)1)

(±95%

CI)

Prote

in0.6

969

189

(±28

417)

10

175

(±4179)

13

054

(±5362)

18

208

(±7478)

3008

(±1235)

5282

(±2169)

1328

(±545)

3459

(±1421)

Lipid

0.7

550

539

(±17

514)

8566

(±2968)

4109

(±1424)

28

077

(±9730)

4723

(±1637)

10

108

(±3503)

3008

(±1043)

3369

(±1168)

Car

bohyd

rate

0.6

320

830

(±10

168)

11

572

(±5923)

33

597

(±16

400)

30

632

(±14

953)

3255

(±1589)

8679

(±4237)

1031

(±503)

1707

(±833)

EPA

0.5

62270

(±1296)

90

800

(±51

844)

12

611

(±7200)

7276

(±4154)

599

(±342)

1645

(±939)

2910

(±1662)

No

EPA

DH

A0.4

9533

(±451)

1209

(±1022)

6662

(±5638)

19

741

(±16

693)

3618

(±3062)

4845

(±4100)

31

352

(±26

529)

No

DH

A

Ener

gy

0.7

40.0

00975

(±0.0

0037)

10

484

(±3925)

6869

(±2607)

26

567

(±9946)

4245

(±1611)

8438

(±3202)

2061

(±782)

3485

(±1322)

DH

A,

doco

sahex

anoic

acid

;EP

A,

eico

sapen

tanoic

acid

.

R. Marshall et al.


needs to be considered when supplying larvae with live

algae. All algal species appear to have sufficient energy

levels to meet the needs of larvae. The species that

requires the highest number of cells is C. calcitrans, at

26 600 larva)1 day)1, but this is hardly surprising given

its relatively small size.

Conclusions

In general, a combination of the algal species I. galbana

and C. calcitrans has been most successful for larval

bivalve rearing. The use of C. calcitrans singly was shown

to yield good results in terms of growth and survival for

C. gigas, V. philippinarum and P. maximus, whereas I. gal-

bana alone did not. The dose–response analysis in this

review may help to explain why this may be the case. The

theoretical maximum effective doses indicated that C. cal-

citrans could supply all of the nutrients necessary to

attain maximum growth rates in C. gigas if the larvae

ingest 30 600 cells larva)1 day)1 (most limited by carbo-

hydrate), a rate that is consistent with the upper limits

provided in the literature (Robert et al. 2001). In contrast,

I. galbana requires a theoretical daily ingestion rate of

90 800 cells larva)1 day)1 for C. gigas to meet the maxi-

mum effective dose levels for all components analysed

(most limited by EPA). This is a very high ration that is

over fourfold higher than the highest reported ration of

T-iso fed to C. gigas (Rico-Villa et al. 2006) and likely to

be well beyond the upper limits of what can actually be

ingested by larvae. Based on the dose–response analysis,

the other species examined can theoretically supply com-

plete nutrition at: 33 600 cells larva)1 day)1 for P. lutheri,

4700 cells larva)1 day)1 for P. tricornutum, 10 100 cells

larva)1 day)1 for S. costatum and 31 400 cells larva)1

day)1 for T. suecica. Like the theoretical levels calculated

for T-iso, these levels exceed the likely upper limits of

what can be ingested in one day by a larva. This is partic-

ularly apparent with the large-sized cells of T. suecica. Its

minimum theoretical ration of 31 400 cells larva)1 day)1

is nearly 10 times higher than the highest reported ration

for T. suecica (3400 cells larva)1 day)1 (Robert et al.

2001)), which indicates that the theoretical ration is

unrealistic. This underscores the advantages of using

multiple species of algae to meet nutritional requirements.

The limitations of this analysis must be recognized,

since it is based on a compilation of standardized data

from experiments that were conducted under different

conditions. Despite using results from various studies, cor-

relations between biochemical components and growth

rates were identified. It must be stressed that the correla-

tions identified in this review do not mean to imply cau-

sality. There may be essential factors (e.g. vitamins, EFAs,

amino acids) that correlate to bulk components in the

algae that are not identified in the literature. In addition,

there are likely to be interactive effects between nutrients

that were not captured in the analysis. Our intent is to

highlight factors that correlate to high growth. Whether

these factors are causative influences on growth should be

the subject of future research.

Future work on the dose–responses of larvae to specific

biochemical components for different bivalve species is

likely to be very helpful. Dose–response information in a

hatchery setting can enable better decision making with

respect to algal species selection, combinations and

rations. An example of how this type of analysis may

prove beneficial is the inclusion of protein in the larval

diet. There is evidence that, as larvae approach settlement,

protein becomes important to survival (Uriarte et al.

2004) and metamorphosis (Utting 1986; Haws et al.

1993). If the requirement for protein increases as meta-

morphosis approaches, there may be benefits to altering

the diet to a higher protein one during late larval stages.

In this case, knowing the appropriate level of protein to

use is important. Research on the dose–response to vari-

ous biochemical components at various stages of larval

growth and development may make it possible for a

hatchery operator to customize the feeds and supplements

used at different times and improve overall growth and

survival rates.

With respect to algal species, bivalve-specific ingestion

rates need further investigation. Pavlova lutheri is a com-

monly used alga in larval bivalve culture and recom-

mended for Crassostrea species in particular (Helm et al.

2004). Investigation of the literature revealed that it is, in

fact, a very poor feed for larvae of C. gigas. It is just one

example of how a commonly used algal species may have

negative effects. This was identifiable in the literature only

because there is a relatively large body of work on

C. gigas. Additional research on species-specific ingestion

and digestion rates will likely reveal similar issues. Studies

of this nature should include not only the ability to ingest

and digest algal species, but also interactions between lar-

val concentrations and cell concentrations and ultimately

how these variables affect ingestion rates.

Some particularly intriguing results were reported by

Gouda et al. (2006). They found that algal species that

were isolated from the native area of P. magellanicus

(Prymnesium sp., N. pelliculosa and C. septentrionalis)

produced superior larval growth and survival compared

with the standard laboratory algae of T-iso, P. lutheri and

C. calcitrans. Although there is a strong justification for

investigating the benefits of using local species and ⁄ or

strains of algae, the desire to undertake a project like this

may be limited owing to the logistics of testing numerous

algal species. In addition, the benefits of using these

native species would need to be very high before they



could be adopted at a commercial hatchery level. Simi-

larly, certain strains of bacteria and picoplankton may

also have benefits (Douillet 1993; Douillet & Landgon

1993; Gallager et al. 1994) for larvae, but the isolation

and testing of countless species may not be desirable or

cost effective.

Research on the use of heterotrophically grown algae

using species such as T. suecica and Schizochytrium sp. as

preserved concentrates or pastes (Coutteau & Sorgeloos

1993) may prove beneficial in the long run. Although not

likely ever to fully replace live algal feeds, their potential

as a supplement or partial replacement has been demon-

strated with C. gigas (Robert et al. 2001) and V. philip-

pinarum (Laing et al. 1990) larvae and could provide

significant cost savings with respect to capital investment

and labour, provided that culture practices are perfected

so that the nutritional value is optimized (Jones et al.

1993; Utting 1993).

One area that requires more research is that of

enriched feeds for larvae. Although PUFA spheres do not

appear to be of much benefit as a single feed source

(Hendriks et al. 2003), microcapsules of chicken ovalbu-

min and fish extracts do seem to be of high value (South-

gate et al. 1992) and may prove suitable for a number of

species. The enrichment of live algal feeds by altering cul-

ture conditions and by adding supplements to the culture

water may also be a very fruitful area of research. Meth-

ods for the modification of protein, TAG and EFAs in

live algae have been established (Utting 1986; Thompson

et al. 1990, 1992; von Elert 2002; Seguineau et al. 2005),

but little research on how these modified feeds affect lar-

val performance has been conducted. Some studies have

shown that modified live feed can improve larval growth

while not negatively affecting survival (Leonardos & Lucas

2000; Pernet et al. 2003). If these methods can be adapted

to the culture of algae in a hatchery, there may be poten-

tial to produce feeds that have a wider spectrum of nutri-

tional components while focusing on algal species that are

readily ingested and digested. In addition, altering the sil-

ica supplement to diatoms may not only increase their

TAG levels, but may also retard test and spine develop-

ment. If this is indeed so, their digestibility could be

improved, making certain diatom species, such as S. cost-

atum and P. tricornutum, more desirable as larval feeds.

Further research is warranted on the enrichment of live

feeds and its effect on larval growth and survival in

numerous bivalve species. Moreover, the direct addition of

dissolved organic components, such as sugars (Welborn

& Manahan 1990), amino acids (Manahan 1983) and

fatty acids (Jaeckle 1995) to larval culture tanks may be a

very direct and simple way to supply essential nutrients

to larvae, although little research has been done in this

area.

Acknowledgements

This review was written during R. Marshall’s PhD

research. R. Marshall was supported financially by a Dis-

covery Grant (to C. M. Pearce) from the Natural Sciences

and Engineering Research Council of Canada and by

Fisheries and Oceans Canada.

References

Albentosa M, Fernandez-Reiriz MJ, Perez-Camacho A, Labarta

U (1999) Growth performance and biochemical composition

of Ruditapes decussatus (L.) spat fed on microalgal and

wheatgerm flour diets. Journal of Experimental Marine Biol-

ogy and Ecology 232: 23–37.

Andersen S, Burnell G, Bergh Ø (2000) Flow-through systems

for culturing great scallop larvae. Aquaculture International

8: 249–257.

Anderson RL, Nelson LA (1975) A family of models involving

intersecting straight lines and concomitant experimental

designs useful in evaluating response to fertilizer nutrients.

Biometrics 31: 303–318.

Anonymous (2006) State of World Aquaculture 2006. FAO

Fisheries Technical Paper No. 500. FAO, Rome.

Babinchak J, Ukeles R (1979) Epifluorescence microscopy, a

technique for the study of feeding in Crassostrea virginica

veliger larvae. Marine Biology 51: 69–76.

Baldwin BS (1995) Selective particle ingestion by oyster larvae

(Crassostrea virginica) feeding on natural seston and cul-

tured algae. Marine Biology 123: 95–107.

Baldwin BS, Newell RIE (1995) Relative importance of differ-

ent size food particles in the natural diet of oyster larvae

(Crassostrea virginica). Marine Ecology Progress Series 120:

135–145.

Bartlett BR (1979) Biochemical changes in the Pacific oyster,

Crassostrea gigas (Thunberg, 1795) during larval develop-

ment and metamorphosis. Proceedings of the National Shell-

fisheries Association 69: 202.

Beattie JH (1992) Geoduck enhancement in Washington

State. Bulletin of the Aquaculture Association of Canada 92:

18–24.

Begg CB, Berlin JA (1988) Publication bias: a problem in

interpreting medical data. Journal of the Royal Statistical

Society 151: 419–463.

Bervera H, Monteforte M (1995) Spat collection trials for pearl

oyster Pinctada mazatlanica at Bahia de La Paz, South Baja

California, Mexico. World Aquaculture Society Book of

Abstracts, p. 62; World Aquaculture 1995, San Diego, Cali-

fornia, USA. February 1–4, 1995.

Brenko MH, Calabrese A (1969) The combined effects of salin-

ity and temperature on larvae of the mussel Mytilus edulis.

Marine Biology 4: 224–226.

Bricelj VM, MacQuarrie SP (2007) Effects of brown tide

(Aureococcus anophagefferens) on hard clam Mercenaria

R. Marshall et al.


mercenaria larvae and implications for benthic recruitment.

Marine Ecology Progress Series 331: 147–159.

Brown MR (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.

Brown MR, McCausland MA (2000) Increasing the growth of

juvenile Pacific oysters Crassostrea gigas by supplementary

feeding with microalgal and dried diets. Aquaculture

Research 31: 671–682.

Brown MR, Robert R (2002) Preparation and assessment of

microalgal concentrates as feeds for larval and juvenile

Pacific oyster (Crassostrea gigas). Aquaculture 207: 289–

309.

Brown MR, Jeffrey SW, Volkman JK, Dunstan GA (1997)

Nutritional properties of microalgae for mariculture. Aqua-

culture 151: 315–331.

Brugere C, Ridler N (2004) Global aquaculture outlook in

the next decades: an analysis of national aquaculture pro-

duction forecasts to 2030. In: FAO Fisheries Circular No.

1001, Fishery Policy and Planning Division 2004, FAO,

Rome.

Bunde TA, Fried M (1978) The uptake of dissolved free fatty

acids from seawater by a marine filter feeder, Crassos-

trea virginica. Comparative Biochemistry and Physiology 60A:

139–144.

Caers M, Coutteau P, Cure K, Morales V, Gajardo G, Sorge-

loos P (1999) The Chilean scallop Argopecten purpuratus

(Lamarck, 1819): II. Manipulation of the fatty acid composi-

tion and lipid content of the eggs via lipid supplementation

of the broodstock diet. Comparative Biochemistry and Physi-

ology 123B: 97–103.

Caers M, Utting SD, Coutteau P, Millican PF, Sorgeloos P

(2002) Impact of the supplementation of a docosahexaenoic

acid-rich emulsion on the reproductive output of oyster

broodstock, Crassostrea gigas. Marine Biology 140: 1157–

1166.

Chavez-Villalba J, Pommier J, Andriamiseza J, Pouvreau S,

Barret J, Cochard J-C et al. (2002) Broodstock conditioning

of the oyster Crassostrea gigas: origin and temperature effect.

Aquaculture 214: 115–130.

Chıcharo L, Chıcharo MA (2001) Effects of environmental

conditions on planktonic abundances, benthic recruitment

and growth rates of the bivalve mollusc Ruditapes decussatus

in a Portuguese coastal lagoon. Fisheries Research 53: 235–

250.

Chu F-L, Greaves J (1991) Metabolism of palmitic, linoleic,

and linolenic acids in adult oysters, Crassostrea virginica.


Chu F-L, Webb KL, Hepworth D, Roberts M (1982)

The acceptability and digestibility of microcapsules by

larvae of Crassostrea virginica. Journal of Shellfish Research

2: 29–34.

Chu F-L, Webb KL, Hepworth D, Casey BB (1987) Metamorph-

osis of larvae of Crassostrea virginica fed microencapsulated

diets. Aquaculture 64: 185–197.

Coutteau P, Sorgeloos P (1993) Substitute diets for live algae

in the intensive rearing of bivalve mollusks – a state of the

art report. World Aquaculture 24: 45–52.

Coutteau P, Hadley NH, Manzi JJ, Sorgeloos P (1994) Effect

of algal ration and substitution of algae by the manipulated

yeast diets on the growth of juvenile Mercenaria mercenaria.


Delaunay F, Marty Y, Moal J, Samain J-F (1993) The effect of

monospecific algal diets on growth and fatty acid composi-

tion of Pecten maximus (L.) larvae. Journal of Experimental

Marine Biology and Ecology 173: 163–179.

Douillet P (1993) Bacterivory in Pacific oyster Crassostrea gigas

larvae. Marine Ecology Progress Series 98: 123–134.

Douillet P, Landgon C (1993) Effects of marine bacteria on

the culture of axenic oyster Crassostrea gigas (Thunberg)

larvae. Biological Bulletin 184: 36–51.

Dunstan GA, Volkman JK, Jeffrey SW, Barret SM (1992) Bio-

chemical composition of microalgae from the green algal

classes Chlorophyceae and Prasinophyceae. 2. Lipid classes

and fatty acids. Journal of Experimental Marine Biology and

Ecology 161: 115–134.

Dunstan GA, Volkman JK, Barrett SM, Leroi J-M, Jeffrey SW

(1994) Essential polyunsaturated fatty acids from 14 species

of diatom (Bacillariophyceae). Phytochemistry 35: 155–161.

Edwards E (2005) Moves to sustainable mussel spats. Fish

Farming International 32 (12): 42.

von Elert E (2002) Determination of limiting polyunsaturated

fatty acids in Daphnia galeata using a new method to enrich

food algae with single fatty acids. Limnology and Oceanogra-

phy 47: 1764–1773.

Enes P, Borges M-T (2003) Evaluation of microalgae and

industrial cheese whey as diets for Tapes decussatus (L.)

seed: effects on water quality, growth, survival, condition

and filtration rate. Aquaculture Research 34: 299–309.

Enright CT, Newkirk GF, Craigie JS, Castell JD (1986) Growth

of juvenile Ostrea edulis L. fed Chaetoceros gracilis Schutt of

varied biochemical composition. Journal of Experimental


Epifanio CE (1979) Comparison of yeast and algal diets for

bivalve mollusks. Aquaculture 16: 187–192.

Ferreiro MJ, Perez-Camacho A, Labarta U, Beiras R, Planas M,

Fernandez-Reiriz MJ (1990) Changes in the biochemical

composition of Ostrea edulis larvae fed on different food

regimes. Marine Biology 106: 395–401.

Gallager SM, Mann R (1981) The effect of varying carbon ⁄nitrogen ratio in the phytoplankter Thalassiosira pseudonana

(3H) on its food value to the bivalve Tapes japonica.


Gallager SM, Mann R (1986) Growth and survival of larvae of

Mercenaria mercenaria (L.) and Crassostrea virginica (Gme-

lin) relative to broodstock conditioning and lipid content of

eggs. Aquaculture 56: 105–121.

Gallager SM, Mann R, Sasaki GC (1986) Lipid as an index

of growth and viability in three species of bivalve larvae.




Gallager SM, Waterbury JB, Stoecker DK (1994) Efficient graz-

ing and utilization of the marine cyanobacterium Synechoco-

cuss sp. by larvae of the bivalve Mercenaria mercenaria.


Garcia EG, Thorarinsdottir GG, Ragnarsson SA (2003) Settle-

ment of bivalve spat on artificial collectors in Eyjafjordur,

North Iceland. Hydrobiologia 503: 131–141.

Gouda R, Kenchington E, Hatcher B, Vercaemer B (2006)

Effects of locally isolated micro-phytoplankton diets on

growth and survival of sea scallop (Placopecten magellanicus)

larvae. Aquaculture 259: 169–180.

Grima EM, Perez JA, Camacho FG, Fernandez FGA, Alonso

DL, Segura del Castillo CI (1994) Preservation of the marine

microalga, Isochrysis galbana: influence on the fatty acid

profile. Aquaculture 123: 377–385.

Haws MC, DiMichele L, Hand SC (1993) Biochemical changes

and mortality during metamorphosis of the Eastern Oyster,

Crassostrea virginica, and the Pacific Oyster, Crassos-

trea gigas. Molecular Marine Biology and Biotechnology 2:

207–217.

Helm MM, Holland DL, Stephenson RR (1973) The effect of

supplementary algal feeding of a hatchery breeding stock of

Ostrea edulis L. on larval vigour. Journal of the Marine Bio-

logical Association of the United Kingdom 53: 673–684.

Helm MM, Bourne N, Lovatelli A (2004) Hatchery Culture of

Bivalves. A Practical Manual. FAO Fisheries Technical Paper

No. 471. FAO, Rome.

Hendriks IE, van Duren LA, Herman PMJ (2003) Effect of die-

tary polyunsaturated fatty acids on reproductive output and

larval growth of bivalves. Journal of Experimental Marine

Biology and Ecology 296: 199–213.

His E, Seaman MNL (1992) Effects of temporary starvation on

the survival, and on subsequent feeding and growth, of oys-

ter (Crassostrea gigas) larvae. Marine Biology 114: 277–279.

His E, Robert R, Dinet A (1989) Combined effects of tempera-

ture and salinity on fed and starved larvae of the Mediterra-

nean mussel Mytilus galloprovincialis and the Japanese oyster

Crassostrea gigas. Marine Biology 100: 455–463.

Holland DL, Spencer BE (1973) Biochemical changes in fed

and starved oysters, Ostrea edulis L., during larval develop-

ment, metamorphosis and early spat growth. Journal of the

Marine Biological Association of the United Kingdom 53: 287–

298.

Jaeckle WB (1995) Transport and metabolism of alanine and

palmitic acid by field-collected larvae of Tedania ignis

(Porifera, Demospongiae): estimated consequences of

limited label translocation. Biological Bulletin 189: 159–167.

Jones DA, Kamarudin MS, Le Vay L (1993) The potential for

replacement of live feeds in larval culture. Journal of the

World Aquaculture Society 24: 199–210.

Jonsson PR, Berntsson KM, Andre C, Wangberg SA (1999)

Larval growth and settlement of the European oyster

(Ostrea edulis) as a function of food quality measured as

fatty acid composition. Marine Biology 134: 559–570.

Laabir M, Amzil Z, Lassus P, Masseret E, Tapilatu Y, De Var-

gas R et al. (2007) Viability, growth and toxicity of Alexand-

rium catenella and Alexandrium minutum (Dynophyceae)

following ingestion and gut passage in the oyster Crassos-

trea gigas. Aquatic Living Resources 20: 51–57.

Labarta U, Fernandez-Reiriz MJ, Perez-Camacho A (1999)

Energy, biochemical substrates and growth in the larval

development, metamorphosis and postlarvae of Ostrea edulis

(L.). Journal of Experimental Marine Biology and Ecology

238: 225–242.

Laing I, Utting SD (1994) The physiology and biochemistry of

diploid and triploid Manila clam (Tapes philippinarum

Adam & Reeve) larvae and juveniles. Journal of Experimental


Laing I, Child AR, Achim J (1990) Nutritional value of dried

algae diets for larvae of Manila clam (Tapes philippinarum).

Journal of the Marine Biological Association of the United

Kingdom 70: 1–12.

Langdon C (1983) Growth studies with bacteria-free oyster

(Crassostrea gigas) larvae fed on semi-defined artificial diets.

Biological Bulletin 164: 227–235.

Langdon C, Waldock MJ (1981) The effect of algal and artifi-

cial diets on the growth and fatty acid composition of Cras-

sostrea gigas spat. Journal of the Marine Biological Association

of the United Kingdom 61: 431–448.

Lavens P, Sorgeloos P (eds) (1996) Manual on the Production

and Use of Live Food for Aquaculture. FAO Fisheries Techni-

cal Paper No. 361. FAO, Rome.

Le Pennec M, Rangel-Davalos C (1985) Observations en

microscopie a epifluorescence de l’ingestion et de la diges-

tion d’algues unicellulaires chez des jeunes larves de Pecten

maximus (Pectinidae, Bivalvia). Aquaculture 47: 39–51.

Leonardos N, Lucas IAN (2000) The nutritional value of algae

grown under different culture conditions for Mytilus edulis

L. larvae. Aquaculture 182: 301–315.

Lombardi AT, Wangersky PJ (1991) Influence of phosphorus

and silicon on lipid class production by the marine diatom

Chaetoceros gracilis growth in turbidostat cage cultures.

Marine Ecology Progress Series 77: 39–77.

Loosanoff VL, Davis HC (1952) Temperature requirements for

the maturation of gonads of the northern oyster. Biological

Bulletin 103: 80–96.

Loosanoff VL, Davis HC (1963) Rearing of bivalve mollusks.

Advances in Marine Biology 1: 1–136.

Lora-Vilchis MC, Maeda-Martinez AN (1997) Ingestion and

digestion index of catarina scallop Argopecten ventricosus-

circularis, Sowerby II, 1842, veliger larvae with ten microal-

gae species. Aquaculture Research 28: 905–910.

Lubet P, Faveris R, Besnard JY, Robbins I, Duval P (1991)

Annual reproductive cycle and recruitment of the scallop

Pecten maximus (Linnaeus, 1758) from the Bay of Seine.

In: Shumway SE, Sandifer PA (eds) Scallop Biology and

Culture, pp. 87–94. The World Aquaculture Society, Baton

Rouge.

R. Marshall et al.


Manahan DT (1983) The uptake and metabolism of dissolved

amino acids by bivalve larvae. Biological Bulletin 164: 236–

250.

Martinez G, Torres M, Uribe E, Dıaz M, Perez H (1992) Bio-

chemical composition of broodstock and early juvenile Chil-

ean scallops, Argopecten purpuratus Lamarck, held in two

different environments. Journal of Shellfish Research 11: 307–

313.

Martınez-Fernandez E, Acosta-Salmon H, Rangel-Davalos C

(2004) Ingestion and digestion of 10 species of microalgae

by winged pearl oyster Pteria sterna (Gould, 1851) larvae.


Martınez-Fernandez E, Acosta-Salmon H, Southgate PC (2006)

The nutritional value of seven species of tropical microalgae

for black-lip pearl oyster (Pinctada margaritifera, L.) larvae.


Milke LM, Bricelj VM, Parrish CC (2008) Biochemical char-

acterization and nutritional value of three Pavlova spp. in

unialgal and mixed diets with Chaetoceros muelleri for

postlarval sea scallops, Placopecten magellanicus. Aquaculture

276: 130–142.

Millar RH, Scott JM (1967) The larva of the oyster Ostrea edu-

lis during starvation. Journal of the Marine Biological Associa-

tion of the United Kingdom 47: 475–484.

Moran AL, Manahan DT (2004) Physiological recovery from

prolonged ‘starvation’ in larvae of the Pacific oyster Crassos-

trea gigas. Journal of Experimental Marine Biology and

Ecology 306: 17–36.

Mourente G, Lubian LM, Odriozola JM (1990) Total fatty acid

composition as a taxonomic index of some marine microal-

gae used as food in marine aquaculture. Hydrobiologia 203:

147–154.

Nell JA, O’Connor WA (1991) The evaluation of fresh algae

and stored algal concentrates as a food source for Sydney

rock oyster, Saccostrea commercialis (Iredale & Roughley),


Nevejan N, Courtens V, Hauva M, Gajardo G, Sorgeloos P

(2003a) Effect of lipid emulsions on production and fatty

acid composition of eggs of the scallop Argopecten purpura-

tus. Marine Biology 143: 327–338.

Nevejan N, Saez I, Gajardo G, Sorgeloos P (2003b) Supple-

mentation of EPA and DHA emulsions to a Dunaliella

tertiolecta diet: effect on growth and lipid composition of

scallop larvae, Argopecten purpuratus (Lamarck, 1819). Aqua-

culture 217: 613–632.

Novoa S, Martınez D, Ojea J, Soudant P, Samain J-F, Moal J

et al. (2002) Ingestion, digestion, and assimilation of

gelatin–acacia microcapsules incorporating deuterium-

labeled arachidonic acid by larvae of the clam Venerupis

pullastra. Journal of Shellfish Research 21: 649–658.

Palacios E, Racotta IS, Kraffe E, Marty Y, Moal J, Samain JF

(2005) Lipid composition of the giant lion’s-paw scallop

(Nodipecten subnodosus) in relation to gametogenesis I. Fatty

acids. Aquaculture 250: 270–282.

Palmer AR (1999) Detecting publication bias in meta-analyses:

a case study of fluctuating asymmetry and sexual selection.

American Naturalist 154: 220–233.

Patil V, Kallqvist T, Olsen E, Vogt G, Gislerød HR (2007)

Fatty acid composition of 12 microalgae for possible use in

aquaculture feed. Aquaculture International 26: 1–9.

Pechenik JA, Lima GM (1984) Relationship between growth,

differentiation and length of larval life for individually

reared larvae of the marine gastropod, Crepidula fornicata.

Biological Bulletin 166: 537–549.

Pernet F, Tremblay R, Bourget E (2003) Biochemical indicator

of sea scallop (Placopecten magellanicus) quality based on

lipid class composition. Part II: larval growth, competency,

and settlement. Journal of Shellfish Research 22: 377–388.

Pernet F, Bricelj VM, Parrish CC (2005) Effect of varying die-

tary levels of x6 polyunsaturated fatty acids during early

ontogeny of the sea scallop Placopecten magellanicus. Journal

of Experimental Marine Biology and Ecology 327: 115–133.

Philippart CJM, van Aken HM, Beukema JJ, Bos AG, Cadee

GC, Dekker R (2003) Climate-related changes in recruit-

ment of the bivalve Macoma balthica. Limnology and Ocean-

ography 48: 2171–2185.

Ponis E, Robert R, Parisi G, Tredici M (2003) Assessment of

the performance of Pacific oyster (Crassostrea gigas) larvae

fed with fresh and preserved Pavlova lutheri concentrates.

Aquaculture International 11: 69–79.

Ponis E, Parisi G, Le Coz JR, Robert R, Zittelli GC, Tredici M

(2006a) Effect of the culture system and culture technique

on biochemical characteristics of Pavlova lutheri and its

nutritional value for Crassostrea gigas larvae. Aquaculture

Nutrition 12: 322–329.

Ponis E, Probert I, Veron B, Le Coz JR, Mathieu M, Robert R

(2006b) Nutritional value of six Pavlovophyceae for Crassos-

trea gigas and Pecten maximus larvae. Aquaculture 254: 544–

553.

Ponis E, Probert I, Veron B, Mathieu M, Robert R (2006c)

New microalgae for the Pacific oyster Crassostrea gigas lar-

vae. Aquaculture 253: 618–627.

Ponis E, Parisi G, Zittelli GC, Lavista F, Robert R, Tredici M

(2008) Pavlova lutheri: production, preservation and use as

food for Crassostrea gigas larvae. Aquaculture 282: 97–103.

Reinfelder JR, Fisher NS (1994) The assimilation of elements

ingested by marine planktonic bivalve larvae. Limnology and

Oceanography 39: 12–20.

Remmenga MD, Milliken GA, Kratzer D, Schwenke JR, Rolka

HR (1997) Estimating the maximum effective dose in a

quantitative dose–response experiment. Journal of Animal

Science 75: 2174–2183.

Rico-Villa B, Le Coz JR, Mingant C, Robert R (2006) Influence

of phytoplankton diet mixtures on microalgae consumption,

larval development and settlement of the Pacific oyster Cras-

sostrea gigas (Thunberg). Aquaculture 256: 377–388.

Rivero-Rodrıguez S, Beaumont AR, Lora-Vilchis MC (2007)

The effect of microalgal diets on growth, biochemical



composition, and fatty acid profile of Crassostrea corteziensis

(Hertlein) juveniles. Aquaculture 263: 199–210.

Robert R, Gerard A (1999) Bivalve hatchery technology: the

current situation for the Pacific oyster Crassostrea gigas and

the scallop Pecten maximus in France. Aquatic Living

Resources 12: 121–130.

Robert R, Miner P, Nicolas JL (1996) Mortality control of scal-

lop larvae in the hatchery. Aquaculture International 4: 305–

313.

Robert R, Parisi G, Rodolfi L, Poli BM, Tredici M (2001) Use

of fresh and preserved Tetraselmis suecica for feeding Cras-

sostrea gigas larvae. Aquaculture 192: 333–346.

Rodrıguez J-L, Sedano FJ, Garcıa-Martın LO, Perez-Camacho

A, Sanchez JL (1990) Energy metabolism of newly settled

Ostrea edulis spat during metamorphosis. Marine Biology

106: 109–111.

Rosenthal R (1979) The ‘‘file drawer problem’’ and tolerance

for null results. Psychological Bulletin 86: 638–641.

Seguineau C, Laschi-Loquerie A, Moal J, Samain JF (1996)

Vitamin requirements in great scallop larvae. Aquaculture

International 4: 315–324.

Seguineau C, Soudant P, Moal J, Delaporte M, Miner P,

Quere C (2005) Techniques for delivery of arachidonic

acid to Pacific oyster, Crassostrea gigas, spat. Lipids 40:

931–939.

Sommer F, Stibor H, Sommer U, Velimirov B (2000) Grazing

by mesozooplankton from Kiel Bight, Baltic Sea, on differ-

ent sized algae and natural seston size fractions. Marine

Ecology Progress Series 199: 43–53.

Soudant P, Paillard C, Choquet G, Lambert C, Reid HI, Marhic

A et al. (2004) Impact of season and rearing site on the phys-

iological and immunological parameters of the Manila clam

Venerupis (= Tapes, = Ruditapes) philippinarum. Aquaculture

229: 401–418.

Southgate PC, Lee PS, Nell JA (1992) Preliminary assessment

of a microencapsulated diet for larval culture of the Sydney

rock oyster, Saccostrea commercialis (Iredale and Roughley).


Strasser M, Hertlein A, Reise K (2001) Differential recruitment

of bivalve species in the northern Wadden Sea after the

severe winter of 1995 ⁄ 96 and of subsequent milder winters.

Helgoland Marine Research 55: 182–189.

Strathmann RR (1967) Estimating the organic carbon content

of phytoplankton from cell volume or plasma volume. Lim-

nology and Oceanography 12: 411–418.

Tammi KA, Soars S, Tumor W, Rice M (1995) Spawning and

spat collection of the bay scallop, Argopecten irradians, in

the Westport River estuary, Massachusetts. World Aquacul-

ture Society Book of Abstracts, p. 223; World Aquaculture

1995, San Diego, California, USA. February 1–4, 1995.

Tan Tiu A, Vaughan D, Chiles T, Bird K (1989) Food value of

eurytopic microalgae to bivalve larvae of Cyrtopleura costata

(Linnaeus, 1758), Crassostrea virginica (Gmelin, 1791), and

Mercenaria mercenaria (Linnaeus, 1758). Journal of Shellfish

Research 8: 399–405.

Tang B, Liu B, Wang G, Zhang T, Xiang J (2006) Effects of

various algal diets and starvation on larval growth and sur-

vival of Meretrix meretrix. Aquaculture 254: 526–533.

Taris N, Ernande B, McCombie H, Boudry P (2006) Pheno-

typic and genetic consequences of size selection at the larval

stage in the Pacific oyster (Crassostrea gigas). Journal of

Experimental Marine Biology and Ecology 333: 147–158.

Thompson PA, Harrison PJ (1992) Effects of monospecific

algal diets of varying biochemical composition on the

growth and survival of Pacific oyster (Crassostrea gigas)

larvae. Marine Biology 113: 645–654.

Thompson PA, Harrison PJ, Whyte JNC (1990) Influence of

irradiance on the fatty acid composition of phytoplankton.

Journal of Phycology 26: 278–288.

Thompson PA, Ming-Xin G, Harrison PJ, Whyte JNC (1992)

Effects of variation in temperature. II: on the fatty acid

composition of eight species of marine phytoplankton. Jour-

nal of Phycology 28: 488–497.

Thorarinsdottir GG (1995) Spat collection and growth of the

Icelandic scallop, Chlamys islandica (O.F. Muller), in sus-

pended culture in Icelandic waters. World Aquaculture Soci-

ety Book of Abstracts, p. 225; World Aquaculture 1995, San

Diego, California, USA. February 1–4, 1995.

Thornton A, Lee P (2000) Publication bias in meta-analysis:

its causes and consequences. Journal of Clinical Epidemiology

53: 207–216.

Torkildsen L, Magnesen T (2004) Hatchery production of scal-

lop larvae (Pecten maximus) – survival in different rearing

systems. Aquaculture International 12: 489–507.

Uriarte I, Farıas A, Hernandez J, Schafer C, Sorgeloos P (2004)

Reproductive conditioning of Chilean scallop (Argopec-

ten purpuratus) and the Pacific oyster (Crassostrea gigas):

effects of enriched diets. Aquaculture 230: 349–357.

Utting SD (1985) Influence of nitrogen availability on the bio-

chemical composition of three unicellular marine algae of

commercial importance. Aquacultural Engineering 4: 175–

190.

Utting SD (1986) A preliminary study on growth of Crassos-

trea gigas larvae and spat in relation to dietary protein.


Utting SD (1993) Procedures for the maintenance and hatch-

ery conditioning of bivalve broodstocks. World Aquaculture

24: 78–82.

Utting SD, Doyou J (1992) The increased utilization of egg

lipid reserves following induction of triploidy in the Manila

clam (Tapes philippinarum). Aquaculture 103: 17–28.

Utting SD, Spencer BE (1991) The Hatchery Culture of Bivalve

Mollusc Larvae and Juveniles. Laboratory Leaflet, Ministry of

Agriculture, Fisheries and Food, Directorate of Fisheries

Research, Lowestoft (68), 31 pp.

Valero JL, Hand C, Orensanz JML, Parma AM, Armstrong D,

Hilborn R (2004) Geoduck (Panopea abrupta) recruitment

in the Pacific Northwest: long-term changes in relation to

climate. California Cooperative Oceanic Fisheries Investiga-

tions Report 45: 80–86.

R. Marshall et al.


Verity PG, Robertson CY, Tronzo CR, Andrews MG, Nelson

JR, Sieracki ME (1992) Relationship between cell volume

and the carbon and nitrogen content of marine photo-

synthetic nanoplankton. Limnology and Oceanography 37:

1434–1446.

Waldock MJ, Holland DL (1984) Fatty acid metabolism in

young oysters, Crassostrea gigas: polyunsaturated fatty acids.

Lipids 19: 332–336.

Webb KL, Chu F-L (1981) Phytoplankton as a food source for

bivalve larvae. In: Pruder GD, Landgon C, Conklin D (eds)

Proceedings of the Second International Conference on Aqua-

culture Nutrition: Biochemical and Physiological Approaches to

Shellfish Nutrition. Special Publication No. 2, pp. 272–291;

Louisiana State University, Baton Rouge, Louisiana, USA.

October 27–29, 1981.

Welborn JR, Manahan DT (1990) Direct measurements of

sugar uptake from seawater into molluscan larvae. Marine

Ecology Progress Series 65: 233–239.

Whyte JNC, Bourne N, Ginther NG (1990) Biochemical and

energy changes during embryogenesis in the rock scallop

Crassadoma gigantea. Marine Biology 106: 239–244.

Whyte JNC, Bourne N, Ginther NG, Hodgeson CA (1992)

Compositional changes in the larva to juvenile develop-

ment of the scallop Crassadoma gigantea (Gray). Journal

of Experimental Marine Biology and Ecology 163: 13–

29.

Yan X, Zhang G, Yang F (2006) Effects of diet, stocking den-

sity and environmental factors on growth, survival, and

metamorphosis of Manila clam Ruditapes philippinarum




Date post:	29-Sep-2016
Category:	Documents
Upload:	robert-marshall
View:	222 times
Download:	1 times

Effects of nutrition on larval growth and survival in bivalves

Documents