Cambios Capacidad Osmoreguladora Tilapia_2010

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Ontogenic changes in the osmoregulatory capacity of the Nile tilapiaOreochromis niloticusand implications for aquaculture

S. Fridman a,, J.E. Bron a, K.J. Rana b

a Institute of Aquaculture, University of Stirling, Scotland, United Kingdomb Aquaculture Division, University of Stellenbosch, South Africa

a b s t r a c ta r t i c l e i n f o

Article history:

Received 24 January 2012Received in revised form 17 April 2012

Accepted 7 May 2012

Available online 12 May 2012

Keywords:

Aquaculture

Brackish water

Osmoregulation

Oreochromis niloticus

Deformity

The Nile tilapia, one of the most extensively cultured tropical freshwater species, offers considerable potential

for culture in low-salinity water. The ontogeny of osmoregulation in the Nile tilapia was studied from

spawning to yolk-sac absorption after exposure to different experimental conditions ranging from freshwater

to 25 ppt. Eggs were able to withstand elevated rearing salinities up to 20 ppt, but transfer to 25 ppt induced

100% mortality by 48 h post-fertilization. Across all stages, embryos and larvae hyper-regulated at lower sa-

linities and hypo-regulated at higher salinities. Osmoregulatory capacity increased during development and

from 2 days post-hatch onwards remained constant until yolk-sac absorption. Adjustments of larval osmolal-

ity following abrupt transfer from freshwater to experimental salinities (12.5 and 20 ppt), followed a pattern

of crisis and regulation, with values for larvae stabilising at c.48 h post-transfer for all treatments, regardless

of age at time of transfer. Age at transfer to experimental salinities (7.520 ppt) had a signicant positive ef-

fect on larval ability to osmoregulate, with larvae transferred at 8 days post-hatch maintaining more constant

whole-body osmolality over the experimental salinities tested than larvae at hatch. Concomitantly, survival

following transfer to experimental salinities increased with age. There was a signicant effect (GLM;

pb0.05) of the salinity of incubation and rearing on the incidence of gross larval malformation that was

seen to decline over the developmental period studied.

2012 Elsevier B.V. All rights reserved.

1. Introduction

In recent times, diminishing freshwater resources resulting from

rapidly increasing urban, industrial and agricultural water use and

from the impact of climate changes, have prompted an urgent need

to manage freshwater, brackish water and marine environments

more efciently. Under these conditions, diversication of aquacul-

tural practices, either by the introduction of new candidate species

or by the adaptation of culture methods for existing species is vital

at a time when innovation and adaptability of the aquaculture indus-

try is fundamental to the maintenance of its sustainability. TheNile ti-

lapia (Oreochromis niloticus,niloticusL.), whose culture now extends

well beyond its natural range, dominates tilapia aquaculture because

of its adaptability and fast growth rate. Although not considered to be

amongst the most salinity-tolerant of the cultured tilapia species, the

Nile tilapia still offers considerable potential for culture in low-

salinity water (Fridman et al., 2012; Suresh and Lin, 1992).

It is well established that measurement of blood or body uid os-

molality in teleosts provides a route for the evaluation of osmoregula-

tory status and the ability to withstand osmotic stress (Alderdice,

1988). Salinity is known to exert selective pressure on studied sh

developmental stages, inuencing reproduction, dispersal and larval

recruitment in marine, coastal and estuarine habitats (Anger, 2003).

Improved knowledge of the limits of salinity tolerance of the Nile

tilapia during the sensitive early life stages and concerning the ability

to predict responses of critical life-history stages to environmental

change could prove invaluable in improving larval rearing techniques

and extend the scope of this globally important sh species, allowing

an expansion of culture area where fresh water is limiting. Recent

reports on ontogenic changes in osmoregulatory capacity during early

life stages have been mainly conned to marine teleost species in

an attempt to explain species and developmental stage-specic distribu-

tioni.e.turbot (Scophthalmus maximus) (Brown and Tytler, 1993), chum

salmon (Oncorhynchus keta) (Kaneko et al., 1995),sea bass(Dicentrarchus

labrax) (Varsamos et al.,2001), Japanese eel (Anquillajaponica) (Okamoto

et al., 2009; Unuma et al., 2005), Mozambique tilapia (Oreochromis

mossambicus) (Yanagie et al., 2009) and the gilt-head sea bream (Sparus

aurata) (Bodinier et al., 2010).

In the present study, the responses to and physiological effects of

osmotic challenge during ontogeny in the Nile tilapia were assessed

through the measurement of embryo and larval osmolality and osmo-

regulatory capacity. In addition, the short-term osmoregulatory re-

sponses of yolk-sac larvae to abrupt transfer to a range of salinities

(7.525 ppt) in terms of osmoregulatory capacity, survival and the

Aquaculture 356357 (2012) 243249

Corresponding author: Tel.: +44 1786 467874; fax: +44 1786 472133.

E-mail address:[email protected](S. Fridman).

0044-8486/$ see front matter 2012 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2012.05.010

Contents lists available at SciVerse ScienceDirect

Aquaculture

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a q u a - o n l i n e
http://dx.doi.org/10.1016/j.aquaculture.2012.05.010http://dx.doi.org/10.1016/j.aquaculture.2012.05.010http://dx.doi.org/10.1016/j.aquaculture.2012.05.010http://dx.doi.org/10.1016/j.aquaculture.2012.05.010http://dx.doi.org/10.1016/j.aquaculture.2012.05.010mailto:[email protected]://dx.doi.org/10.1016/j.aquaculture.2012.05.010http://www.sciencedirect.com/science/journal/00448486http://www.sciencedirect.com/science/journal/00448486http://dx.doi.org/10.1016/j.aquaculture.2012.05.010mailto:[email protected]://dx.doi.org/10.1016/j.aquaculture.2012.05.010


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related incidence of deformity were investigated. This is the rst

study to give a complete picture of the ontogeny of osmoregulatory

ability over a range of salinities during successive early life stages in

the euryhaline Nile tilapia and provides valuable insights into

ontogenic variations in the capacity of this species to hyper- and

hypo-regulate over a range of salinities.

2. Materials and methods

2.1. Egg supply, preparation of media and rearing systems

All eggs were obtained from Nile tilapia breeding populations

held at the Tropical Aquarium, Institute of Aquaculture, University of

Stirling, UK. Broodstock were maintained individually in partitioned

200 l freshwateraquaria with re-circulated, pre-conditioned freshwater

(local tap water aerated and heated to 281 C for 24 h prior to use)

and fed on articialpellets (#5 trout pellet, Trouw Aquaculture Limited,

Skretting, UK). The light regime was maintained at a 12:12 h light:dark

period. Eggs were obtained from ripe females by manually stripping

eggs from a ripe female into a Petri dish followed by the addition of

freshly-stripped milt from two males per female. After 12 min fertili-

zation, water was added and eggs were placed in their respective incu-

bation unit and designated as a batch.

Independent test incubation units each comprised a 20 l plastic

aquarium with an attached Eheim pump (Series 94051) supplying

61 L plastic bottles using down-wellingow (Rana, 1988). Tempera-

ture in the incubation units was maintained at 28 1 C using individ-

ual 300 W thermostatically controlled heaters (Visi-therm, Aquarium-

systems, Mentor, Ohio, USA).The experimental salinities were prepared

using conditioned water (tap water aerated and heated to 28 1 C for

24 h prior to use) and commercial salt (Tropic Marin, Aquarientechnic,

D-36367, Germany) and salinity was measured using a salinity refrac-

tometer (Instant Ocean Hydrometer, Marineland Labs., USA) accurate

to 1 ppt. About 10% of water was replaced daily in the incubation

aquaria to compensate for evaporation and salinity was adjusted ac-

cordingly. Dead eggs and larvae were removed daily to prevent fungal

infection. The light regime was maintained as for broodstock.

2.2. Ontogenic prole of osmoregulatory capacity

In the rst experiment, ovarian uid and pre-fertilized eggs

were initially sampled for osmolality. Eggs were then fertilized in

freshwater and transferred at 34 hours (h) post-fertilization to

the experimental salinities i.e. 7.5, 12.5, 17.5, 20 and 25 ppt. Control

eggs remained in freshwater. Sampling was initially performed at

time of transfer and subsequently at developmental points during

embryogenesisi.e.gastrula (c.24 h post-fertilization) and completion

of segmentation period (c. 48 h post-fertilization) and then at hatch,

2, 4 and 6 days post-hatch andnally at yolk-sac absorption. Triplicate

experiments were conducted using three different batches of eggs,

with each batch sub-divided between three replicate round-bottomed

incubators within each incubation unit. A pooled sample of 30 embryosor newly hatchedyolk-sac larvae (10 from each replicate) wascollected

at each sampling point, rinsed in distilled water (DW), blotted dry and

immediately frozen at 70 C.

The small size of Nile tilapia embryos and yolk-sac larvae prevented

efcient collection of blood or specic body uids for osmolality

measurements therefore whole-body measurements were used for

osmolality measurements. This approach was validated by initialexper-

iments that measured the effect of yolk materials on osmolality values

in which the osmolalities of larval body regions were measured and

no signicant differences were found between them. Pools of whole

larvaewere thawed on ice, homogenised with a motorisedTeon pestle

(Pellet Pestle Motor,Kontes) andthe homogenate centrifuged at 10 C

for 10 min at 14000 g (Eppendorf centrifuge, 5417R). The supernatant

overlying the pellet was carefully removed into a single well of a

96-well plate and thoroughly mixed with a pipette to ensure homoge-

neity of each sample. Osmolality was determined using an Advanced

3MO Plus MicroOsmometer (Advanced Instruments, MA, USA.) using

three replicates of 20l aliquots of supernatant from each pool and

the accuracy of the machine was regularly checked against calibration

standards of 50 and 850 mOsm kg1 and osmolality was expressed as

whole body osmolality (mOsmol kg1).

2.3. Acclimation time

In the second experiment, the acclimation time of yolk-sac larvae

at hatch, 3 and 6 days post-hatch to abrupt salinity challenge was

assessed to determine the time necessary for whole-body osmolality

to reach a steady-state after abrupt transfer from the rearing medium

(freshwater) to two experimentalsalinities(12.5 and 20 ppt). Triplicate

experiments were conducted using three different batches of eggs.

Pooled samples, consisting of 30 whole yolk-sac larvae collected at

1.5, 3, 6, 12, 24, 48 and 72 h post-transfer, were rinsed in distilled

water (DW), blotted dry and immediately frozen at 70 C. Whole

body osmolality (mOsmol kg1) was determined as described above.

2.4. Osmoregulation and survival following abrupt salinity challenge

In a third experiment, healthy yolk-sac larvae were transferred

directly from freshwater to 7.5, 12.5, 17.5 or 25 ppt at hatch, 2, 4,

6 and 8 days post-hatch. Larvae were exposed to their experimental

salinity for 48 h prior to sampling. Control larvae remained in fresh-

water. Triplicate experiments were conducted using three different

batches of eggs. Pooled samples, consisting of 30 whole yolk-sac larvae

(10 from each replicate) were rinsed in distilled water (DW), blotted

dry and immediately frozen at 70 C. Whole body osmolality

(mOsmol kg1) was determined as described above.

2.5. Larval malformation

Thirty newly-hatched yolk-sac larvae from each of the three

batches from the rst experiment were selected at random from

freshwater, 12.5 and 20 ppt and were examined under a dissectingmicroscope in order to observe incidence and type of malformations.

Thereafter, thirty live yolk-sac larvae were selected at regular time

points during yolk-sac absorption i.e.2, 4, 6 days post-hatch and com-

plete yolk-sac absorption and malformations were assessed as before.

The percentage of abnormality was calculated, based on the numbers

of normal and malformed larvae as follows: percentage of malformed

larvae (%)=100number of malformed larvae/number of normal

larvae.

2.6. Statistics

Statistical analyses were carried out with Minitab 16 using a

General Linear Model (GLM) or with Tukey's post-hoc pair-wise

comparisons (pb0.05). Homogeneity of variance was tested usingLevene's test and normality was tested using the AndersonDarling

test. Where data failed assumptions, they were transformed using an

appropriate transformation. All percentage data were normalised by

arcsine-root transformation prior to statistical analyses and data are

presented as back-transformed mean and upper and lower 95% con-

dence limits. Signicance was accepted when b0.05 and results were

expressed as mean S.E.

3. Results

3.1. Ontogenic prole of osmoregulatory capacity

Osmolalityof unfertilized eggs (358.24.95 mOsmol kg1) was sim-

ilar to that of ovarian uid (370.7 2.30 mOsmol kg1

) but was seen to

244 S. Fridman et al. / Aquaculture 356357 (2012) 243249


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drop signicantly (GLM with Tukey's post-hoc pair-wise comparisons;b0.05) to 216.9 8.89 mOsmol kg1 after 34 h post-fertilization in

freshwater (Fig. 1). There was always a signicantly higher whole body

osmolality in eggs and larvae maintained in elevated salinities as

compared to those in freshwater. Osmolality during embryogenesis in

freshwater dropped further to a low of 174.6 4.15 mOsmol kg1 at

completion of the segmentation period at c. 48 h post-fertilization, and

then was seen to increase signicantly (GLM; b0.05) by hatching to

230.3 2.53 mOsmol kg

1

. Osmolality of larvae in freshwater was thenseen to rise again signicantly (GLM; pb0.05) by 4 days post-hatch

and, thereafter, maintained a relatively constant level of 319.54.91

324.8 7.41 mOsmol kg1 until yolk-sac absorption (Fig. 1). In contrast,

the osmolality of eggs transferred to elevated salinities at 34 h

post-fertilization increased with increasing salinity immediately

upon transfer. Transfer to 25 ppt induced 100% mortality by 48 h

post-fertilization. In the higher salinities of 17.5 and 20 ppt,

osmolality was seen, after the initial abrupt rise, to steadily increase,

reaching a maximal value of 434.0 2.07 mOsmol kg1 and 497.8

2.79 mOsmol kg1 at hatch for larvae maintained in 17.5 and 20 ppt

respectively, declining signicantly (GLM; b0.05) at 2 days post-

hatch and thereafter maintaining a relatively constant level until yolk-

sac absorption(Fig. 1). For the lower salinities of 7.5 and 15 ppt, follow-

ing a similar abrupt rise at transfer, osmolality appears to drop slightly

at c. 48 h post-fertilization and then rise signicantly (GLM; b0.05)

by 4 days post-hatch, similarly maintaining a relatively constant level

thereafter until yolk-sac absorption.

3.2. Salinity tolerance

The time required for whole-body osmolality to stabilise following

an abrupt transfer to an elevated salinity did not appear to vary

according to age at transfer (Fig. 2). In general, the changes in osmo-

lality appeared to follow a pattern of a rise in values followed by a

slight decline and stabilisation, with values for larvae stabilising at c.

24 h for all treatments, regardless of age at time of transfer, and

subsequently remaining the same with no signicant change (one-

way ANOVA; b0.05) until 72 h post-transfer. According to these re-

sults, the subsequent experiments on osmolality were made on larvae

havingreached a steady-state osmolality following48 h exposure to ex-

perimental salinities.

Ontogeny had a signicant (GLM; b0.05) effect on larval ability to

withstand abrupt osmotic challenge; larvae at 8 days post-hatch

maintained a more constant osmolality over the experimental salinities

tested (range 341.4 11.06 to 427.0 2.34 mOsmol kg1) than larvae

transferred at hatch (360.93.33 to 487.7 4.92 mOsmol kg1

)(Fig. 3).

Survival generally was high and decreased with increasing salinity

but increased with successive developmental stages (Table 1). Survival

Fig. 3. Whole-body osmolality following 48 h after transfer to elevated salinities. Mean

S.E.; different letters represent signicant differences between treatments at a given age of

transfer (General Linear Model with Tukey'spost-hocpairwise comparisons;b

0.05).

Fig. 1. Ontogenic changes in whole body osmolality of Nile tilapia larvae. MeanS.E.;

different numerals indicate signicant difference between treatments at selected sampling

points (General Linear Model with Tukey's post-hocpair-wise comparisons;b

0.05).

Fig. 2. Time-course changes in whole body osmolality in Nile tilapia larvae following

direct transfer from freshwater to 12.5 and 20 ppt at hatch, 3 days post-hatch and

6 days post-hatch. Mean S.E.

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rates of 98% were recorded for larvae maintained in freshwaterat hatch

yet lower survival rates, in the rangeof 8392%, were recorded for those

transferred, at hatch, to elevated salinities. Larvae transferred to salin-

ities of 7.517.5 ppt at 2 and 4 days post-hatch exhibited an improved

survival rate with respect to that at hatch, yet larvae transferred to

20 ppt still displayed a signi

cantly lower survival rate (GLM; b

0.05)than other salinities. From 6 days post-hatch onwards, no signicant

differences were observed between survival rates amongst salinities

(GLM;b0.05) (Table 1).

3.3. Incidence of malformation

Gross larval malformation was dened in terms of visible peri-cardial oedema, sub-epithelial oedema of the yolk-sac, non-specic

Fig. 4. Malformation during yolk-sac absorption periodin Nile tilapia. A) Normal larvae at hatch in freshwater showing network of blood vesselsassociatedwith yolk-sac syncytium.

B) Malformed larvae at hatch maintained in 17.5 ppt showing curvature of stunted tail and pericardial haemorrhaging (arrowhead). C) 2 days post-hatch larvae maintained in

20 ppt showing pericardial oedema (arrow) and haemorrhaging of blood vessels associated with the yolk-sac syncytium (arrowhead). D) 2 days post-hatch larvae maintained

in 20 ppt with pericardial oedema, enlarged heart (arrow) and sub-epithelium oedema of the yolk-sac (arrowhead). E) Normally developing larvae at yolk-sac absorption

maintained in freshwater. F) 8 days post-hatch larvae maintained in 20 ppt showing distortion of neurocranium (arrowhead) and pooling of blood along spine (arrow).

Table 1

Effect of various salinities on larval survival (%) at 48 h post-transfer at various developmental stages during yolk-sac absorption period. Mean and 95% condence limits. Different

superscript letters represent signicant differences between treatments; different subscript letters represent signicant differences between age at transfer (General Linear Model

with Tukey'spost-hocpairwise comparisons; b0.05).

Larval survival (%)

Salinity: Freshwater 7.5 ppt 12.5 ppt 17.5 ppt 20 ppt

Time of transfer:

Hatch 98

(94.49.9)aab

86

(70.696.4)ab

92

(82.997.8)aab

83

(73.790.2)ac

85

(70.795.6)abc

2 dph 98

(95.499.9)aa

98

(94.599.9)ba

95

(79.599.9)aa

97

(93.599.6)ba

85

(69.495.9)ab

4 dph 99

(98.499.9)aa

96

(90.799.5)ba

92

(86.296.9)aa

95

(90.198.8)ba

77

(69.684.2)ab

6 dph 99

(95.199.8)aa

98

(94.899.9)ba

96

(87.199.9)aa

95

(85.199.7)ba

99

(95.599.3)ba

8 dph 99

(96.899.9)aa

99

(94.899.9)ba

97

(90.899.9)aa

99

(96.899.9)ba

99

(96.899.9)ba

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haemorrhaging of blood vessels associated with the yolk-sac syncytium

and body or the appearance of an abnormal neurocranium (Fig. 4). Inci-

dence of malformation of yolk-sac larvae was always signicantly

higher at elevated salinities than in freshwater at all stages (GLM;

pb0.05). Incidence of malformation was seen to decline signicantly

(GLM; pb0.05) from hatch until yolk-sac absorption (Table 2).

4. Discussion

4.1. Ontogenic pattern of osmoregulatory capacity and salinity tolerance

This study is the rst to consider the ontogeny of osmoregulatory

capacity in a tilapiine species over a range of salinities. Whilst osmo-

lality levels of embryos initially showed a rapid rise following transfer

to hyper-osmotic environments, embryos still displayed some sort of

regulative control, with the exception of embryos transferred to

25 ppt, which were unable to survive. This would suggest that embry-

os maintain some sort of osmoregulatory control during these early

stages of embryogenesis. At spawning the yolk is enclosed by a dou-

ble membrane enclosing a thin layer of cytoplasm which concentrates

on the animal pole forming a blastodisc. During gastrulation the pe-

ripheral cells of the morula begin to cover the yolk sac, coinciding

with the appearance of cutaneous mitochondria-rich cells (MRCs)

i.e.on the epithelium of the body surface and yolk-sac of the develop-

ing embryo, thus marking the start of the selective restriction of ions

and water transfer or active ionoregulation (Guggino, 1980). The rst

appearance of MRCs on the yolk-sac epithelium of dechorionated

freshwater maintained Mozambique tilapia (O. mossambicus) embryos

was reported at 26 h post-fertilization but no apical crypt suggesting

functionality was found until 48 h post-fertilization (Lin et al., 1999).

Similarly, Ayson et al. (1994)observed MRCs on theyolk-sacepithelium

of the Mozambique tilapia (O. mossambicus) embryos at 30 h post-

fertilization in both freshwater and seawater, but apical openings of

MRCs were rst observed at a low density at 48 h post-fertilization or

half-way to hatching. The presence of functional MRCs, therefore, may

offer an explanation for the ability of embryos, as demonstrated in

this study, to maintain osmotic controli.e.to hyper-regulate in low sa-

linity waters (i.e.freshwater and 7.5 ppt) and to hypo-regulate in ele-vated salinities (i.e. 12.520 ppt) at 48 h post-fertilization following

completion of epiboly.

The current study reports an increase in osmolality levels for all

treatments upon hatching. These results are contrary to previously pub-

lished results in marine teleost species where the regulative ability of

larvae at hatch to maintain osmoregulatory control through measure-

ment of body-uid concentration has been reportede.g.Pacic salmon

spp.(Weisbart, 1968), herring (Clupea harengus)(Holliday and Blaxter,

1960), plaice (Pleuronectes platessa)(Holliday and Jones, 1965, 1967),

Atlantic halibut (Hippoglossus hippoglossus) (Riis-Vestrgaard, 1982;

Hahnenkamp et al., 1993) and turbot (S. maximus)(Brown and Tytler,

1993). The decline in osmoregulatory capacity described in the present

study upon hatch is therefore surprising. This could be explained

by an osmoregulatory challenge dueto thelossof theprotective cho-

rion at hatch. In a previous study,Fridman et al. (2012)reported lar-

val mortalities immediately upon hatch for Nile tilapia larvae

incubated and hatched in varying experimental salinities (i.e.0, 7.5,

15, 20 and 25 ppt) which declined by 3 days post-hatch and levelledout by 5 days post-hatch, suggesting that Nile tilapia face the

greatest osmoregulatory challenge immediately after hatching, yet

show an increasing capacity to maintain ionic and osmotic balance

that is conferred ontogenically through the yolk-sac period. Indeed

results from the present study illustrate that, once hatching occurs,

osmolality levels begin to move towards a more constant range

from 4 days post-hatch until yolk-sac absorption for all larvae both in

freshwater and elevated salinities (7.520 ppt) suggesting an improve-

ment in ability to osmoregulate as larvae develop. Indeed, Yanagie et al.

(2009)reports a similar maintenance of osmolality at a relatively con-

stant level for yolk-sacO. mossambicus larvae maintained in freshwater

from hatch until yolk-sac absorption at around 320 mOsmol kg1. It is

well documented that teleost yolk-sac larvae are able to maintain os-

motic and ionic gradients between their internal and external environ-

ments, due mainly to the presence of numerous extrabranchial

mitochondria-rich cells (MRC) commonly observed on the abdominal

epithelium of the yolk-sac and other body surfaces ofsh larvae. A dis-

tinct spatial shift in MRC distribution from body surface to branchial

areas during ontogeny is acknowledged and has been reportedin sever-

al speciesi.e.the European sea bass (D. labrax) (Varsamos et al., 2002),

the killish (Fundulus hereroclitus) (Katoh et al., 2000), the Mozam-

bique tilapia (O. mossambicus)(van der Heijden et al., 1999; Yanagie

et al., 2009) and the Nile tilapia (O. niloticus)(Fridman et al., 2011).

The short-term iono-regulatory responses of yolk-sac larvae to

abrupt transfer to elevated salinities (7.520 ppt) were assessed. Pre-

liminary trials for estimation of adaptation time in this study, as de-

ned by whole-body osmolality measurements, showed larvae at all

stages displaying a rise in osmolality followed by recovery period to

reach a steady state after 48 h post-transfer. Results in other specieshave shown a similar pattern in body-uid osmolality e.g. Mozam-

bique tilapia (O. mossambicus) 48 h post-transfer from freshwater to

26 ppt for osmolality levels to reach original levels (Hwang and Wu,

1993), sea bass (D. labrax) a crisis and recovery period for 27 days lar-

vaeis reported after abrupt transfer from 25 pptto 39.5 ppt and 5.3 ppt

to reach a steady state by 48 h post-transfer (Varsamos et al., 2001) and

juvenile red drum (Sciaenops ocellatus) a stabilisation in blood osmolal-

ity levels occurs following direct transfer from sea water to freshwater

after 96 h (Crocker et al., 1983).

Ontogenic changes in salinity tolerance appear, in this study, to be

related to developmental stage. Results suggest that abrupt osmotic

challenge gave rise to different osmoregulatory responses which were

dependant on the ontogenic stage of the larvae and, moreover, a gradu-

al improvement in ability to osmoregulate occurs during ontogeny. Thisability to maintain osmotic homeostasis is reected in survival patterns

of larvae following transfer; from 6 days post-hatch onwards, with no

signicant difference being evident in survival between salinities after

this point. The study byWatanabe et al. (1985)on the ontogeny of

salinity tolerance in various tilapiine spp. (e.g. Oreochromis aureus,O. niloticus and O. mossambicusO. niloticus hybrid) spawned and

reared in freshwater but transferred to elevated salinities (032 ppt)

from 7 to 120 days post-hatch suggested that changes in salinity toler-

ance were more closely related to body size than chronological age,

and were probably related to maturational eventssuch as the functional

development of the osmoregulatory system. Although thesh in that

study were older than those used in the present study, it is still interest-

ing to note that ontogenic physiological changes may confer osmoregu-

latory ability and salinity tolerance.

Table 2

Effect of salinity on larval malformation during yolk-sac absorption period. Mean and

95% condence limits were calculated on arcsine square transformed data. Different

superscript letters represent signicant differences between treatments; different sub-script letters represent signicant differences between days (General Linear Model

with Tukey'spost-hocpairwise comparisons; b0.05).

Incidence of malformation (%)

Salinity: Freshwater 12.5 ppt 20 ppt

Time of transfer:

Hatch 14

(1259.6)aa

22

(20.641.9)ab

23

(19.932)ab

2 dph 2

(0.517.6)ba

8

(6.234.8)abb

29

(22.635.6)ac

4 dph 2

(0.44.7)ba

8

(2.2318.1)abb

10

(2.423.6)bb

6 dph 1

(0.115.1)ba

2

(0.115.1)ba

6

(1.913.6)bb

Yolk-sac absorption 1

(0.56.0)ba

7

(5.68.5)abb

9

(7.511.7)bb



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4.2. Larval malformation

In the current study, there was a signicant negative effect (GLM;b0.05) of increasing salinity on the occurrence of larval malformations

during the yolk-sac period. A high incidence of larval abnormalities has

been previously reported during early life stages of marine teleosts,

when challenged with variations in salinity. Larvae of the navaga

(Eleginus nava), polar cod (Boreofadus saida) and Arctic ounder

(Liopsetta glacialis) exhibited a high incidence of malformation in lowsalinities (Doroshev and Aronovich, 1974), as did the Atlantic halibut

(H. hippoglossus) (Bolla and Ottesen, 1998). A lower percentage of

abnormalities in the newly hatched larvae of the pomfret (Pampus

punctatissimus) was reported at 2930 ppt than either at b25 ppt

or >40 ppt (Shi et al., 2008) and, similarly, the percentage of deformi-

ties was signicantly lower at 36 ppt than at either lower (2433 ppt)

or higher (3642 ppt) salinities in the Japanese eel (A. japonica)

(Okamoto et al., 2009). These results suggest that, once the incubation

and rearing salinity moves away from that which is encountered in

nature, detrimental effects become more pronounced, a trend that

is apparent in the current study.

It is clear therefore that there also exists a signicant effect of ontog-

eny on the incidence of malformation during the yolk-sac period. The

development of the branchial system and an ontogenic shift in location

of active MRCs fromextrabranchial to branchial sites is widely accepted

and has been reported in the Mozambique tilapia (O. mossambicus) by

Li et al. (1995)andvan der Heijden et al. (1997)and in the Nile tilapia

(O. niloticus) (Fridman et al., 2011) which would appear to confer an in-

creasing osmoregulatory capacity which is apparent in the reported

pattern of survival in elevated salinities following hatch. This observed

reduction of pericardial and sub-epithelial oedema as yolk-sac larvae

develop appearsto reect an increasing ability to maintain ionic and os-

motic balance throughout the yolk-sac period. In agreement, oedema is

not observed in zebrash larvae (Danio rerio) after exposure to contam-

inants if exposure is delayed during ontogeny suggesting that larvae are

particularly vulnerable shortly after hatching (Belair et al., 2001).

Haemorrhaging and pooling of blood appears to be linked to

oedematous build up during yolk-sac stages of the Nile tilapia. It is

possible that oedema may compress the delicate blood capillary net-work on the yolk-sac syncytium, and have a damaging, systemic ef-

fect on whole larvae by impairing circulation. Hill et al. (2003)

examined the negative impacts of contaminant polychlorinated

dibenzo-p-dioxins (PCDDs) on the epithelium of zebrash (D. rerio)

during early life stages and reported a build-up of oedema and ensu-

ing organ compression that led to decreased kidney and circulatory

function. They concluded that this model also predicts that many dif-

ferent types of stresses, withinwhich salinity must be included, might

lead to the same outcome, and this therefore offers a possible expla-

nation for the observations made in this study.

4.3. Implication for aquaculture

It has become increasingly clear in recent years that, given our -nite resources, long-term sustainability of aquaculture must be based

on an efcient use of natural resources. The early phase of the life

cycle in teleost shes is usually thought of as the most crucial period

due to the poorly developed regulatory system i.e.gills and kidneys

and the rapidly occurring developmental changes (Ozoh, 1979). In

the current study, the assessment of whole body osmolality has pro-

vided a method that has allowed an evaluation of the osmoregulatory

status during the early life stages of the Nile tilapia. These measure-

ments appear to offer valuable insight into the emerging pattern of

the adaptive capacity to hypo- and hyper-regulate during ontogeny.

Osmolality levels of embryos immediately post-transfer to elevat-

ed salinities appear to be proportional to and directly related to the

osmolality of the external medium, but then drop to a more steady

state during embryogenesis and yolk-sac period. This suggests that

in these latter stages an ontogenic capacity for regulatory control de-

velops, which in turn affects the ability of larvae to withstand transfer

to elevated salinities and decreases the incidence of larval deformity.

Data regarding the ontogeny of osmoregulation and adaptive strate-

gies of the Nile tilapia, a commercially important teleost sh, provide

valuable tools for predicting timing of occurrence of adaptive ability

and improving larval rearing techniques. Additionally, an increase in

knowledge of the limits and basis of salinity tolerance of Nile tilapia

during the particularly sensitive early life stages and the ability topredict responses of critical life-history stages to environmental

change could prove invaluable, extending the scope of this globally

importantsh species.

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