Effect of salinity on egg hatching, yolk sac absorptionand larval rearing of Senegalese sole (Solea senegalensisKaup 1858)Emilio A. Salas Leiton, Ana Rodriguez-Rua, Esther Asensio, Carlos Infante, Manuel Manchado,Catalina Fernandez-Dıaz and Jose P. Canavate

IFAPA Centro ‘El Toruno’, Junta de Andalucıa, El Puerto de Santa Marıa, Cadiz, Spain

Introduction

Adjusting culture salinity to optimum physiological

requirements is needed to fulfil the aquaculture develop-

ment of any species. The influence of salinity is usually

variable over the productive cycle, requiring specific stud-

ies on the different life stages (Varsamos et al. 2005). The

effect of salinity during the egg incubation period and

subsequent hatching has been studied in various aquacul-

ture species, including sparids such as silver sea bream

(Sparus sarba) (Mihelakakis & Kitajima 1994) and Japa-

nese red sea bream (Pagrus major) (Apostolopoulos

1976), serranids such as Dicentrarchus labrax (Marangos

et al. 1986) and mugilids such as stripped mullet (Mugil

cephalus) (Walsh et al. 1991). Similar studies have been

carried out in turbot (Scophthalmus maximus) (Kuhlmann

& Quantz 1980; Karas & Klingsheim 1997; Nissling et al.

2006), greenback flounder (Rhombosolea tapirina) (Hart

& Purser 1995) and Brazilian flounder (Paralichthys orbi-

gnyanus) (Sampaio et al. 2007), representatives of scoph-

thalmids, pleuronectids and paralichthyds, respectively. In

soleids, existing information is restricted to the common

sole (Solea solea); studies by Fonds (1979) and Devau-

chelle et al. (1987) analysed the effect of salinity, in com-

bination with temperature, on egg development and later

hatching rates. Studies on early overall development,

Correspondence

Emilio A. Salas Leiton, IFAPA Centro

‘El Toruno’, Junta de Andalucıa, Apartado 16,

11500 Puerto de Santa Marıa, Cadiz, Spain.

Email: emilioa.salas@juntadeandalucia.es

Received 19 October 2011; accepted

23 January 2012.

Abstract

Assays carried out under laboratory-controlled conditions revealed that eggs of

Senegalese sole, Solea senegalensis Kaup 1858, incubated at 10, 18, 27 and 33

grams per litre (g L)1) reached their maximum hatching rates (above 80%)

48 h after incubation. In contrast, hatching was significantly delayed (24 h)

and completely failed when the salinity was set at 5 and 0 g L)1, respectively.

Newly hatched and early-developing yolk sac larvae presented similar survival

rates 3 days after hatching (DAH) when exposed to salinities of 10, 18, 27 and

33 g L)1. Larvae incubated at 5 g L)1 salinity died a few hours after hatching

once they were released from their respective chorions. The notochord length

was unmodified by salinity 5 DAH. Nevertheless, an overall lower myotomal

height and a putative delay in the depletion of yolk sac were found in larvae

reared at 10 g L)1. This anomaly in overall larval development became more

pronounced with the occurrence (100% of analysed larvae) of mouth deformi-

ties and a lack of functionality under 10 g L)1 salinity. Transferring 2 DAH

larvae from 33 g L)1 (exhibiting normal mouth development) to a medium

with a salinity of 10 g L)1 had no effect on first feeding, weight and metamor-

phic index. Overall our results indicate the existence of a key time window

(0–2 DAH) in which a salinity value higher than 10 g L)1 is required to

achieve adequate mouth development and further functionality of larvae. The

present work represents a valuable contribution to the sole industry, opening

new possibilities in hatchery management practices.

Key words: deformity, egg hatching, growth, salinity, Solea senegalensis, yolk sac larvae.

Reviews in Aquaculture (2012) 4, 49–58 doi: 10.1111/j.1753-5131.2012.01060.x

ª 2012 Blackwell Publishing Asia Pty Ltd 49

survival, yolk sac absorption, biometric measurements

and malformations have evaluated the salinity tolerances

of marine larvae of pelagic species such as Atlantic red

porgy (Pagrus pagrus) (Ostrowski et al. 2011), Japanese

eel (Anguilla japonica) (Okamoto et al. 2009), leopard

grouper (Mycteroperca rosacea) (Gracia-Lopez et al. 2004)

and mangrove red snapper (Lutjanus argentimaculatus)

(Estudillo et al. 2000). In flatfish, knowledge is limited to

newly hatched Atlantic halibut (Hippoglossus hippoglossus

L.) larvae (Lein et al. 1997). No information about the

influence of salinity during the egg incubation period and

in yolk sac absorbing larvae is available for Senegalese

sole. This type of basic information is of high value for

the sole industry to optimise the conditions required for

larval rearing.

The relationship between salinity and growth through-

out the larval stages, once the mouth opening process has

been completed and the first food is supplied, has been

demonstrated to be highly species dependent. In flatfish,

both Brazilian and greenback flounders showed optimum

growth over salinities ranging from 20 to 30 g L)1 (Sam-

paio et al. 2007) and from 15 to 35 g L)1, respectively

(Hart et al. 1996). The spotted halibut (Verasper variega-

tus) preferred moderately low salinities (8–16 g L)1) dur-

ing the larva–juvenile transformation period (Wada et al.

2004). In contrast, larvae of southern flounder (Paralich-

tys lethostigma) showed reduced survival and markedly

lower growth at full-strength seawater (35 g L)1) com-

pared with that achieved at a lower salinity (25 g L)1)

from hatching to 15 days after hatching (DAH) (Mousta-

kas et al. 2004). The possibility of successfully rearing sole

larvae under reduced salinity conditions would lead to

new options for hatchery facilities where only brackish

water is available.

Senegalese sole is a marine teleost that uses estuarine

areas as nursery grounds (Cabral 2000; Vinagre et al.

2009; Ramos et al. 2010). Its natural ability to inhabit

environments where the salinity varies significantly has

allowed traditional rearing under extensive conditions

in the coastal wetlands of the Iberian Peninsula (Drake

et al. 1984). Over the past decade, this flatfish has been

proposed as a high potential species to diversify South

Atlantic and Mediterranean intensive aquaculture (Dinis

et al. 1999; Imsland et al. 2003). Although salinity is

presumed to play a pivotal role in the life cycle of

Senegalese sole, the only currently available information

about the potential effects of salinity is limited to

growth (Arjona et al. 2009) and the osmoregulatory

response (Arjona et al. 2007) of juvenile soles (37–

44 g). The present study describes the influence of dif-

ferent salinities on the egg hatching rate and yolk sac

larval stage. Tolerance to low salinity in terms of

growth, first food ingestion, metamorphic index and

survival is also evaluated under standard larval rearing

procedures.

Materials and methods

Egg hatching rate assay

Eggs were obtained from a wild origin Senegalese sole

broodstock stocked in Centro IFAPA ‘El Toruno’ (Puerto

de Santa Marıa, Cadiz, Spain) and kept in a flow-through

system under a naturally fluctuating temperature regime.

The detailed feeding protocol and basic physicochemical

rearing parameters of this type of breeding husbandry

have been previously reported (Anguıs & Canavate 2005).

Eggs were removed from egg collectors to avoid mixing

of successive spawns and their quality was evaluated by

examining the proportion of buoyant eggs. Spawns exhib-

iting proportions above 70% were used in the experi-

ments (Kjørsvik et al. 2003). The synchrony of embryo

development in any spawn was confirmed in the labora-

tory after binocular observation.

Eggs at the blastodisc stage with no embryo develop-

ment, end of gastrular overgrowth and blastopore closed,

equivalent to stage 1 or 3–4 according to Fonds (1979) or

Devauchelle et al. (1987), respectively, from a single natu-

ral spawning (Fonds 1979) were used. The hatching rate of

eggs incubated in freshwater (0 g L)1) and at five different

salinity levels (5, 10, 18, 27 and 33 g L)1) was registered

daily. Salinities lower than 33 g L)1 were achieved by dilu-

tion with distilled water. Sixty eggs in total were deposited

in 12 well plates (4 mL well)1 as working volume and

5 eggs well)1). Plates were incubated at 20�C under full

darkness for 72 h. Each salinity level had triplicate plates.

Egg hatching rates (EHR) (Eqn 1) related to every salinity

were calculated at 24, 48 and 72 h after egg incubation:

EHR (%) ¼ ðNHL=NIEÞ � 100 ð1Þ

where NHL is the number of hatched larvae and NIE is

the number of incubated eggs.

Effect of salinity on early developing yolk sac larvae

Eggs from additional spawnings were incubated as

described above in media with salinity values of 5, 10, 18,

27 and 33 g L)1 to evaluate the effect of salinity on

survival, development and feeding incidence in yolk sac

larvae. Recently hatched larvae were transferred to 12

well plates (4 mL well)1 as working volume; n = 3 lar-

vae well)1) under the same salinity conditions. Larvae

were kept without any food at 20�C at a light intensity of

200 lx from 0 to 5 DAH. The water volume (75%) was

exchanged every 48 h. Each salinity level (5, 10, 18, 27

and 33 g L)1) had triplicate plates. Larval survival (LS)

E. A. Salas Leiton et al.

Reviews in Aquaculture (2012) 4, 49–5850 ª 2012 Blackwell Publishing Asia Pty Ltd

(Eqn 2) up to 5 DAH was calculated from daily registered

mortality:

LS (%) ¼ ðNAL=NHLÞ � 100 ð2Þ

where NAL is the number of alive larvae and NHL is the

number of hatched larvae.

Notochord length, myotomal height and yolk sac vol-

ume (YSV) were the biometric indicators used to evaluate

overall early development. The mouth opening process

and the occurrence of deformities were also recorded

from microscope image capture using a digital camera.

Notochord length and myotomal height were measured

using an eyepiece micrometer under a light microscope.

Yolk sac volume was calculated from the major and

minor axes of the yolk sac, considering the formula for

an ellipsoidal mass (Eqn 3) (Yufera et al. 1999). The

lengths were measured by IMAGE J v. 1.44 software

(National Institute of Health, Bethesda, Maryland, USA)

(Abramoff et al. 2004):

YSV (nL) ¼ ðp=6Þ � L�H2 ð3Þ

where L is the length of the major axis of the yolk sac

(nm) and H is the length of the minor axis of the yolk

sac (nm).

The feeding incidence was assessed in 5 DAH larvae after

checking the proportion of larvae that had prey items in

their stomachs. These larvae in the well plates were offered

rotifers (Brachionus plicatilis) at a concentration of 15 roti-

fers mL)1. Larvae were then collected after 2 h and their

stomachs analysed under a microscope to determine the

presence of rotifers. The percentage of larvae with positive

food ingestion was obtained for each salinity value.

Standard larval rearing at low salinity

Fertilized eggs from a single spawning were initially

stocked in cylinder ⁄ conical tanks (800–1000 eggs L)1) at

system salinity (33 g L)1). Incubation was developed in

flow-through water (one renewal per h) using gentle aera-

tion and a temperature of 20�C. At 2 DAH, after check-

ing that the mouths of the larvae had opened adequately,

the larvae were transferred to 900 L cylinder larval rearing

tanks. Two different salinities were assayed in duplicate

tanks: 10 and 33 g L)1. An initial density of 40 larvae L)1

(working volume of 200 L in each tank) and 16 h

light : 8 h dark photoperiod (intensity of 600–800 lx) was

set. A daily 20% water exchange cultivation system was

established. Essential physicochemical rearing parameters

were controlled daily through routine measurements

at 09.00 h. The mean (± sd) water temperature was

21.5 ± 0.02�C and dissolved oxygen levels were kept at

6.9 ± 0.04 mg L)1 (equivalent to 84.5 ± 1.15% gas-

saturation). Final mean salinities were 10.7 ± 0.02 and

34.2 ± 0.13 for the 10 and 33 g L)1 experimental condi-

tions, respectively. The applied feeding protocol consisted

of 15 rotifers mL)1 from 2 to 4 DAH, 20 rotifers mL)1

from 5 to 8 DAH, 10 metanauplii of Artemia sp. per mL

from 9 to 13 DAH, 15 metanauplii mL)1 from 14 to 17

DAH and 20 metanauplii mL)1 from 18 to 21 DAH. Both

live preys were previously enriched with Isochrisis gal-

bana, clon T-ISO. Lyophilized Nannochloropsis gaditana

(1 mg L)1) was added to the tanks from 2 to 8 DAH to

enhance background and rotifer nutritional status. Bot-

tom debris was siphoned out when needed and neither

chemical nor antibiotic treatments were used at any time.

First ingestion activity as a function of salinity was

examined at 2, 4 and 6 DAH following the methodology

described above. Sole larvae were sampled on 2, 4, 6, 8,

11, 14, 18 and 21 DAH for growth measurements. These

larvae were placed on pre-tared weighing filters. The fil-

ters were then dried at 60�C for 48 h and weighed to the

nearest 0.1 mg to calculate individual dry weight. Meta-

morphic development in relation to salinity was evaluated

through an index ranging from 0 to 4 based on the eye

migration process (Fernandez-Diaz et al. 2001). Feeding

activity, dry weight and the metamorphic index were

studied in triplicate samples (n = 25 per sample). The lar-

val survival percentage was calculated once the experi-

ment was finished (21 DAH).

Statistical analysis

Differences in mean egg hatching rates between the tested

salinities were determined by one-way anova once nor-

mality and homocedasticity were verified. Normality and

similarity in the variances were checked using Kolmo-

gorov–Smirnov test and F-Fisher contrast, respectively.

P-values higher than 0.10 and 0.05 were accepted as

significant in the respective analyses.

Significant statistically differences (P < 0.05) in survival

rate, development indicative biometric parameters (noto-

chord length, myotomal height and yolk sac volume) and

first ingestion tests in yolk sac larvae were determined by

one-way anova followed by post-hoc Fisher’s Least

Significance Difference (LSD) multiple comparison tests.

If the data violated the assumptions of normality and

homocedasticity required for a one-way anova, a

Kruskal–Wallis non-parametric test was used.

Student’s t-tests were used to compare differences in

dry weight, metamorphic index and survival rate between

10 and 33 g L)1 salinities on every sampling day through-

out the larval rearing period. Differences were considered

significant when P < 0.05. Normality and homocedas-

ticity were again confirmed before using the Student’s

t-tests.

Salinity effects on Solea senegalensis

Reviews in Aquaculture (2012) 4, 49–58ª 2012 Blackwell Publishing Asia Pty Ltd 51

Results

Influence of salinity on egg hatching

No hatching differences among the four highest tested

salinities (10, 18, 27 and 33 g L)1) were identified

(P > 0.05) 24 h after egg incubation (Fig. 1). Eggs main-

tained under these salinities reached their maximum

hatching rates (above 80%) after 48 h, with only the

33 g L)1 experimental condition leading to significantly

lower hatching. Significant differences were only found

between the 18 and 33 g L)1 media (hatching rates of

95.7% and 80.7%, respectively) after completing the incu-

bation period (72 h) (Fig. 1). It is important to note that

eggs exposed to a 5 g L)1 salinity medium showed a rele-

vant hatching delay (P < 0.05), a clearly observable effect

24 and 48 h after incubation (Fig. 1). Embryonic develop-

ment of eggs in the 0 g L)1 group was interrupted during

the first 48 h. Because the hatching rate was close to zero

at any time, this group has not been included in Figure 1.

The exceptional embryos that did survive (<5%) to break

their respective chorions died just at the time of contact

with fresh water.

Early development of yolk sac larvae in relation to

salinity

Significant differences in the survival rate of yolk sac lar-

vae at different salinities were found from 1 to 3 DAH

(Fig. 2). Because all of the larvae in the 5 g L)1 salinity

medium died a few hours after being released from their

chorions, this condition has not be included in Figure 2

(further developmental analyses and feeding tests could

not be carried out). Exposure to low and medium salini-

ties (10, 18 and 27 g L)1) led to higher survival compared

with 33 g L)1 salinity at 1 and 2 DAH. One day later

(3 DAH), this difference (P < 0.05) was only detected

between the 10 and 33 g L)1 rearing mediums. Survival

rates decreased progressively after 4 DAH and significant

differences between assayed salinities were not found

when the experiment finished (5 DAH) (Fig. 2). Owing

to the absence of any adequate mobility or performance

indicator in the larvae that survived under 10 g L)1 salin-

ity at 5 DAH, a beating heart was the adopted criteria to

consider the larvae alive.

A relevant progressive increasing of notochord length

from hatching to 3 DAH was observed in larvae from

every experimental group. Although higher lengths at

10 g L)1 (P < 0.05) could be observed at 1 and 2 DAH,

no significant effect caused by salinity on larval length

was obtained when the assay concluded (Fig. 3a). Overall

evolution of myotomal heights did not show differences

between 18, 27 and 33 g L)1 salinities throughout the

assay. Lower heights in contrast were measured in larvae

kept at a salinity of 10 g L)1; these differences were par-

ticularly significant at intermediate days (1–4 DAH)

(Fig. 3b). Low salinity (10 g L)1) also favoured increased

volumes in the yolk sac; this result was significant at 1–2

DAH. The main difference was in the time taken to fully

deplete the yolk sac, larvae at 10 g L)1 required one more

day (3 DAH) than larvae at medium and high salinities

(2 DAH) (Figure 3c).

The influence of salinity on early development became

more evident when mouth morphology was examined.

Every larva cultivated at low salinity (10 g L)1) had a

non-functional mouth. Undeveloped jaws, abnormal

lower jaw development and the absence of an upper jaw

or gaping jaws were the most common recorded deformi-

ties (Fig. 4). No deformed jaws were observed in larvae

incubated in the higher salinity media. The feeding

Figure 1 Hatching rates (%) (mean ± SE) of eggs incubated at salin-

ities of 5, 10, 18, 27 and 33 g L)1. Different letters indicate significant

differences (P < 0.05) between the groups.

Figure 2 Survival rate (%) (mean ± SE) of yolk sac larvae at salinities

of 10, 18, 27 and 33 g L)1. Different letters indicate significant differ-

ences (P < 0.05) between the groups.

E. A. Salas Leiton et al.

Reviews in Aquaculture (2012) 4, 49–5852 ª 2012 Blackwell Publishing Asia Pty Ltd

activity test carried out with 5 DAH larvae confirmed that

larvae grown at 10 g L)1 salinity were incapable of feed-

ing. Although 60–82% of larvae exposed to salinities of

18, 27 and 33 g L)1 were able to ingest food, no rotifers

were observed in the stomachs of the larvae when the

salinity was 10 g L)1 (Fig. 3d).

(a) (b)

(c)(d)

Figure 3 (a) Notochord lengths (mm), (b) myotomal heights (mm) and (c) yolk sac volumes (nL) [mean ± standard deviation (SD)] in larvae reared

at salinities of 10, 18, 27 and 33 during the first 5 days after hatching (DAH). The proportion of 5 DAH larvae ingesting rotifers (mean ± SD) is

indicated in (d). Significant different subgroups are denoted by * and + (a,b,d). In (c), * indicates significant differences between 10 g L)1 and the

remaining salinities and ) denotes differences (P < 0.05) between 10 and 33 g L)1 salinities.

(a) (b)

(c) (d)

Figure 4 (a) Normally developed larva found at 18, 27 and 33 g L)1 salinities. (b) Undeveloped jaw, (c) absent upper jaw and (d) gaping jaw

mouth deformities observed in larvae reared at 10 g L)1 salinity.

Salinity effects on Solea senegalensis

Reviews in Aquaculture (2012) 4, 49–58ª 2012 Blackwell Publishing Asia Pty Ltd 53

Effects of reducing seawater salinity on larval rearing

following standard procedures

The first food ingestion test after transference to larval

rearing tanks (2 DAH) did not reveal any differences

(P > 0.05) between larvae kept at 10 and 33 g L)1 salini-

ties on any sampling day (Fig. 5). A positive tendency in

the proportion of larvae ingesting food could be observed

when the stomach contents of the larvae were analysed

on 2, 4 and 6 DAH (Fig. 5). Both salinities led to similar

dry weights throughout the experimental period with lar-

vae reared at 10 g L)1 weighing significantly more only at

21 DAH (Fig. 6a). Final weights were 988.6 ± 106.3

(mean ± standard deviation) and 799.9 ± 11.9 lg (21

DAH) after 19 days for larvae cultivated at salinities of 10

and 33 g L)1, respectively.

An analysis of metamorphosis revealed that both exper-

imental groups started this process between eight and 11

DAH (Fig. 6b). Although larvae reared at 33 g L)1 salinity

presented a significantly higher index in the middle of

metamorphosis (14 DAH), the slower development of lar-

vae grown at low salinity could be finally compensated

when metamorphosis concluded (metamorphic index of

four at 21 DAH) (Fig. 6b). With regard to larval survi-

val, a significantly higher rate was obtained at 33 g L)1

salinity after 21 days of standard rearing. Although

63.3 ± 8.4% (mean ± standard deviation) of larvae sur-

vived in tanks under the higher salinity at the end of

experimental period, 44.9 ± 0.9% survived at 10 g L)1

salinity.

Discussion

High hatching rates (above 80%) were obtained when

incubating eggs for 48 h at salinities of 10, 18, 27 and

33 g L)1. Of special relevance was the finding that a salin-

ity of 5 g L)1 induced a 24 h delay in the hatching

embryos and that no egg hatching was obtained in the

0 g L)1 group. Although an overall wide salinity range for

successful egg development is a shared feature in estua-

rine-dependent flatfish, the optimum salinity for hatching

might be considered to be species dependent. No effect of

salinity on hatching rate was found in eggs of greenback

flounder incubated over the range 15–45 g L)1 (Hart &

Purser 1995). In Brazilian flounder, higher egg hatching

rates were obtained at increased salinity (the assayed

range was 5–35 g L)1), coinciding with positive egg buoy-

ancy (Sampaio et al. 2007). Such a result may, however,

lead to confusion because inadequate water quality rather

than an effect of salinity per se was responsible in that

study for declining egg-hatching rates at low salinities. A

changing optimum salinity has been found for hatching

of turbot eggs, as a consequence of selective adaptations

to local environmental conditions (Nissling et al. 2006).

Although stocks of turbot in the Baltic Sea had maximum

hatching rates at salinities ranging from 7 to 15 g L)1

(Nissling et al. 2006) and those in the Belt Sea were

restricted to 15 g L)1 (Kuhlmann & Quantz 1980), turbot

Figure 5 Analysis of larval feeding activity (%) (mean ± standard

deviation) based on the presence or absence of rotifers in the stom-

ach. Feeding response at 2, 4 and 6 days after hatching.

(a) (b)

Figure 6 (a) Dry weight (lg) and (b) metamorphic index evolutions (mean ± standard deviation) of larvae reared under standard conditions at

salinities of 10 and 33 g L)1 over 21 days. *P < 0.05.

E. A. Salas Leiton et al.

Reviews in Aquaculture (2012) 4, 49–5854 ª 2012 Blackwell Publishing Asia Pty Ltd

inhabiting the North Sea had optimum rates at salinities

above 20 g L)1 (Karas & Klingsheim 1997). In the com-

mon sole, Fonds (1979) study pointed out that eggs of

this species, which is closely related to the Senegalese sole,

might be successfully developed until hatching over the

range 10–40 g L)1. Devauchelle et al. (1987) confirmed

that 20–35 g L)1 was the optimum salinity range for

hatching in S. solea and that decreased or even non-

existent egg hatching rates were obtained at salinities

below 10 g L)1. Our results obtained for Senegalese sole

are in accordance with those described for common sole

(Fonds 1979; Devauchelle et al. 1987). An important

point to note in the present work is the significant hatch-

ing delay following incubation at 5 g L)1 and the failure

to hatch at 0 g L)1. Low temperature (Kazuyuki et al.

1988), low pH (Oyen et al. 1991) and hypoxia (Wu 2009)

have been reported as environmental conditions inducing

retarded embryonic development. The highly controlled

laboratory conditions under which our assay was carried

out enable us to discard external conditions as the reason

for the differences in hatching times, pointing directly to

salinity as the only responsible factor. Osmolality retarded

embryonic development has been described in freshwater

species eggs incubated in brackish media (Kinne & Kinne

1962; Yang & Chen 2006). To the best of our knowledge,

the present study is the first to report an egg-hatching

delay induced by low salinity in marine flatfish. Embryos

of teleost fish possess an extremely low overall permeabil-

ity (Mangor-Jensen 1987). Nevertheless, the ability to

osmoregulate during the early development stages (e.g.

blastula cells) was demonstrated in Atlantic herring

(Clupea harengus) (Holliday & Jones 1965) and plaice

(Pleuronectes platessa) (Holliday & Jones 1967). Moreover,

eggs of the freshwater North American minnow (Hybo-

gnathus amarus, Cyprinidae) adapted their specific gravity

and volume to the surrounding salinity, revealing an

osmotic flux of water out from the periviteline space into

the incubation medium (Cowley et al. 2009). The mas-

sively aborted embryonic development found in our

0 g L)1 group indicates that early developing eggs were

affected by the extremely low hypo-osmotic medium and

that a certain chorion permeability to the external med-

ium can be inferred (Rawson et al. 2000). The hatching

delay induced in our 5 g L)1 group remains difficult to

understand. In this regard, the extra energy mobilization

associated with osmoregulation in eggs of Takifugu obscu-

rus (Yang & Chen 2006) could be considered.

Yolk sac larvae revealed no survival differences from 4

DAH in relation to salinities over the range 10–33 g L)1.

This high tolerance is in agreement with results obtained

for leopard grouper (0–40 g L)1) (Gracia-Lopez et al.

2004) and in opposition to the narrower ranges described

in species such as halibut (27–32 g L)1) (Lein et al. 1997)

and eel (24–36 g L)1) (Okamoto et al. 2009). Although

every group examined presented similar notochord

lengths from 3 DAH, the myotomal height of yolk sac

larvae grown at 10 g L)1 salinity was significantly lower.

Larger yolk sacs and an apparent delay in their consump-

tion were also found in that group. One noticeable find-

ing was the diverse jaw abnormalities (affecting 100% of

larvae) and the subsequent ingestion inability associated

with a salinity of 10 g L)1. As described in Pagrus pagrus

(Ostrowski et al. 2011), notochord length was not affected

by salinity. A similar inverse relationship between yolk sac

volume and salinity in sole has been previously described

in larvae of diverse species such as Atlantic herring

(Holliday & Blaxter 1960), Pacific herring (Clupea pallası)

(Alderdice & Velsen 1971), pomfret (Pampus punctatissi-

mus) (Shi et al. 2008) and halibut (Lein et al. 1997).

Although a delay in yolk consumption affecting overall

development could be inferred from this relationship, sev-

eral authors have proposed higher water content, as a

consequence of osmotic disequilibrium, to be responsible

for the increased sac volume under reduced salinity con-

ditions (May 1974; Lein et al. 1997; Shi et al. 2008). The

lower myotomal height measured in larvae reared at

10 g L)1 salinity might also suggest differential energy

mobilization associated with salinity; although such larvae

would demand more energy to cope with an osmotically

unfavourable external environment (Lein et al. 1997; Shi

et al. 2008), the remaining salinity conditions (18, 27 and

33 g L)1) would allow them to make more efficient use

of the yolk resources to build body tissues. These general

developmental anomalies are supported by the exclusive

occurrence of jaw deformities in larvae reared at 10 g L)1

salinity. Nutritional unbalance (Gisbert et al. 2008) and

environmental conditions such as pollution (Sun et al.

2009), extreme temperature (Okamura et al. 2007) and

physical stress (Morrison & MacDonald 1995) have been

reported as likely causes of jaw deformities. Knowledge

about abnormalities in jaw development, and the result-

ing inability to feed (Morrison & MacDonald 1995),

induced by salinity is, however, more limited. Permanent

gaping jaws syndrome has been characterized in Atlantic

halibut larvae incubated at salinities £34 g L)1 (Lein et al.

1997), whereas eel (Okamoto et al. 2009) and Pacific

herring (Alderdice & Velsen 1971) suffered delayed jaw

development, abnormal lower jaws and gaping jaws in

£33 and £25 g L)1 salinity mediums, respectively. As the

rearing water dilution was the only differential condition

existing between our experimental groups, deficiencies in

certain trace ions (e.g. zinc) (Somasundaram et al. 1984)

might be considered to be the origin of the jaw malfor-

mations found in sole larvae raised at low salinity. Addi-

tional studies must be conducted to confirm this

hypothesis.

Salinity effects on Solea senegalensis

Reviews in Aquaculture (2012) 4, 49–58ª 2012 Blackwell Publishing Asia Pty Ltd 55

Larval rearing until complete metamorphosis under

standard culture conditions demonstrated that sole larvae

could be successfully reared at 10 g L)1 if transference to

this low salinity is carried out once the mouth opening

process has been completed (2–3 DAH). No significant

differences were found between salinities of 10 and

33 g L)1 when feeding activity was analysed in the first six

rearing days. Both salinities led to similar individual dry

weights throughout the experimental period, with signifi-

cantly higher weights recorded in larvae grown at 10 g L)1

salinity only at 21 DAH. With the exception of 14 DAH,

salinity did not induce differences in the development of

metamorphosis, a process completed 21 DAH under both

conditions. A higher final survival was, in contrast,

obtained in larvae cultivated at 33 g L)1 salinity. This

overall high tolerance to salinity shown by sole larvae is in

opposition to that reported for other relevant flatfish such

as Brazilian flounder and spotted halibut. Both of these

species presented narrower ranges of salinity (20–30 g L)1

and 8–16 g L)1, respectively) for successful growth during

larval rearing (Wada et al. 2004; Sampaio et al. 2007). The

ability of Senegalese sole larvae to grow over a wide salin-

ity range following standard husbandry is similar to that

reported for greenback flounder (15–35 g L)1) (Hart et al.

1996). Nevertheless, lower survival rates were obtained in

greenback flounder larvae reared at 15 g L)1 salinity in

comparison with either 25 or 35 g L)1. The higher weight

achieved at 21 DAH in our larvae reared at 10 g L)1 salin-

ity is probably associated with decreased survival, and

consequently with lower stocking densities during the last

days of metamorphosis. An inverse relationship between

growth parameters and the stocking density of flatfish is

well known both in larvae (Bolasina et al. 2006) and early

juveniles (Irwin et al. 1999; Schram et al. 2006; Merino

et al. 2007). Despite the fact that the causes of increased

mortality in low salinity groups might be diverse, an

adequate hydrodynamic design of the tanks, which com-

pensates for the higher relative weight of larvae placed in

10 g L)1 rearing waters, is a key issue that must be devel-

oped. Our overall results demonstrate that Senegalese sole

larvae have the capacity to be successfully reared following

standard procedures at salinities as low as 10 g L)1, with

the only requisite being completed mouth development

before transference to low salinity. That is, larvae can be

kept in incubators at medium or high salinity (‡18 g L)1)

until 2 DAH and then transferred to rearing tanks.

In conclusion, the present work demonstrated an

absence of differences in Senegalese sole egg hatching over

a salinity range of 10–33 g L)1. Anatomical abnormalities

during early development, mainly mouth deformities,

occurred when larvae were hatched and subsequently kept

at 10 g L)1 salinity. The results reveal a key time window

(0–2 DAH) where a salinity higher than 10 g L)1 is

required for adequate mouth development and further

functionality of larvae. Investigations to determine the

effect of low salinity on metabolic and osmotic stress

routes in Senegalese sole larvae are recommended.

Acknowledgements

This work was financed by next projects: INVESOLEA

(CC08-33) and RTA2009-00066-00-00 from the ‘Instituto

Nacional de Investigacion y Tecnologıa Agraria y Alimen-

taria’ (INIA, Spain) and FEDER (UE) funds, and the

AQUAGENET (SOE2 ⁄ 1381P1 ⁄ E287) – INTERREG IVB

SUDOE program. Dr Rodrıguez-Rua was supported by

an IFAPA postdoctoral contract co-financed by the Euro-

pean Social Fund (2007–2013).

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