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Salinity stress response in shrimp postlarvae:
Elena Palacios and Ilie S. Racotta CIBNOR, Mexico30
Aniversario
Relation to further performance and physiological basis
Salinity stress test applied to PL• The salinity stress test (SST) is commonly applied to
shrimp postlarvae (PL) to determine their quality or physiological condition.
• PL are abruptly exposed to low salinities, and then survival is determined after 30 m to 2 h. Batches with high survival (above 60%) are considered of better quality.
Postlarvae
Seawater
1 L containers
Diluted sea water
30-50 PL/replica
Recount of live PL
SST is based on the life cycle of shrimp:At PL stage, shrimp invade estuaries, where they are exposed to abrupt changes in salinity.
SST and further performance• Higher survival to SST is associated to greater
survival during stockinga and better performance (growth and survival) during growoutb, but this has not been tested in controlled conditions
aFegan 1992; bAquacop et al. 1991
• This is surprising, given the general belief that SST is predictive of performance to stocking and growout.
• The SST was first proposed by Tackaert et al. (1989), but its use as a predictive criterion has been limited to correlations for survival between PL stages (PL10 to PL15a ; PL1 to PL20b)
aRees et al. 1994; bRacotta et al. 2004
Our first questions were: • Is survival to SST correlated to:
– survival during stocking?– performance during growout?
SST and further performance
SST PL15n = 40 batches(x triplicates n=50 PL)
Growout: in 1m2 cement tanks (nurseries) 2 months n = 40 batches
n = 60 Batches (Individual spawns) reared separately from N to PL
Stocking: in buckets Cumulative Mortalities10 days, PL were not fedn = 40 batches
Alvarez et al. 2004
• Batches with higher survival to a SST also had higher survival during stocking (r=0.34).
Alvarez et al. 2004
• Survival to SST was not correlated to performance during growout.
The SST can be used, under some circumstances, to determine PL quality or physiological condition, on a short term, such as stocking
• What does a “better physiological condition” mean?
• What are we evaluating when using a SST?
Taken from Bouaricha et al. 1994
“…there is a strong correlations between salinity tolerance, the development of osmoregulatory epithelia, the ability to osmoregulate, and activity of Na+/K+-ATPase pump”
Taken from Charmantier et al. 1988
The tolerance of PL to changes in salinity is associated to increased osmoregulatory capacities
…. “salinity tolerance and osmoregulation appear correlated”…reaching a maximum at PL10-12
In accordance, when comparing SST in different studies, the older the PL, the higher the survival.For review: Racotta et al. 2003
Péqueux 1995 (crabs)
Towle & Weihrauch, 2001 (crabs)
Osmoregulatory models for crabs...
Morris, 2001(marine and terrestrial crabs)
• When crabs are placed in diluted seawater, there is an influx ofwater and a loss of ions.
• In osmoregulating crabs, the Na+ ions are pumped into the hemolymph by the Na+/ K+-ATPase pump located in the basolateral membrane.
In shrimp PL…
Alvarez et al. 2004
• In accordance, the activity of Na+/K+-ATPase pump was higher in PL15 exposed to lower salinities.
• However, the Na+/K+- ATPase activity was not significantly higher in batches with higher survival to a SST.
0123456789
30 psu 5 psu
Na+
/K+-
AT
Pase
act
ivity
(uM
P/h
)
High survival SST Low survival SST
A
B
In shrimp PL…
Survival to a SST was correlated to PL gill area (r=0.55), even when corrected for size
Alvarez et al. 2004
Survival to a SST was correlated to size of PL (r=0.62), even in PL of the same age
• In the absence of perceptible differences e.g. treatment, survival to a SST is affected by development (size and gill area), even in PL of the same age.
• Maybe differences between same-age PL, such as quantity of accumulated reserves, can affect osmoregulatory mechanisms and survival to salinity changes?
Palacios et al. 2004a
Fed (3 hrs before sampling)
Starved (24 hrs before sampling)
SST PL20
SST PL20
Sampling
Sampling
SST and energy reservesAssuming that the higher the levels of energy reserves the PL has, the better its physiological condition:
• Does this improve osmoregulatory mechanisms? • Is this reflected on the survival to a SST?
n = 6 batches(x triplicates n=50 PL20)
To test this, a SST was applied to 24 h starved PL and to fed PL.
SST and energy reserves
TG in hepatopancreas were lower in:• starved PL in control salinity • fed and starved PL exposed to low salinity.
In gills, TG were higher in:• Fed PL exposed to low salinity• followed by starved PL exposed to low salinity.
0
1
2
3
4
5
30 psu 5 psu
TG
(mg/
g)
Fed PL Starved PL
a
b b b
Palacios et al. 2004a
0.0
0.2
0.4
0.6
0.8
1.0
30 psu 5 psu
TG
(mg/
g)
Fed PL Starved PL
a
b b
ab
• Could this be the result of transfer of lipids from hepatopancreas to gills?
• Fed PL had more reserves to provide energy for overall response to salinity stress and/or Na+/K+-ATPase pump
SST and energy reservesAs expected, survival to a SST was higher in fed PL.
• The Na+/K+-ATPase activity was higher in fed PL at the control salinity (baseline), indicating that it was affected by quantity of energy reserves.
• However, no differences were found for Na+/K+-ATPase activity when PL were exposed to low salinities.
More reserves did result in better survival, but survival to a SST cannot be fully explained by decreased Na+/K+- ATPase pump activity74
76
78
80
82
84
86
88
Surv
ival
SST
(%)
Fed PL Starved PL
a
Palacios et al. 2004a
0
20
40
60
80
100
120
140
160
30 psu 5 psu
Na+
/K+-
AT
Pase
(µM
P/g
/h)
Fed PL Starved PL
a
bc c
b
The QUANTITY of energy reserves is not associated to Na+/K+- ATPase pump activity, as the main osmoregulatory mechanism.
What about the QUALITY of reserves?
HUFA, or highly unsaturated fatty acids levels in the diet, have been one of the most tested variables in relation to SST.
Higher survivals to a SST and during rearing have been reported in PL offered HUFA. However, it is not clear if HUFA can affect directly osmoregulatory mechanisms…
SST and HUFA • Can HUFA-rich diets modify gill membrane
lipids?• If so, can they modulate osmoregulatory
mechanisms?To test this, PL1 were fed Artemia nauplii enriched with three ICES emulsions x 20 d
Low HUFA (0%) SST PL20
SamplingMedium HUFA (30%)
High HUFA (50%) n = 9 batches(x triplicates n=50 PL20)
Palacios et al. 2004b
SST and HUFA
Palacios et al. 2004b
• DHA was the only fatty acidaffected by salinity, with lower levels in PL exposed to low salinity
0
5
10
15
20
25
30
35
40
30 psu 5 psu
HU
FA (%
)
Low HUFA Medium HUFA High HUFA
• HUFA-enrichment affected fatty acids in the membrane phospholipids of gills.
• No effect was observed in total HUFA, or most of the other fatty acids, in membrane gills of PL exposed to low salinity
0
5
10
15
20
25
30 psu 5 psu
DH
A (%
)
AB
aab b
bb
SST and HUFA
Palacios et al. 2004b
• No significant differences were found for rearing performance (survival and growth) between HUFA-enriched diets.
0
20
40
60
80
100
Rearing survival (%) Weight (mg)
Low HUFA Medium HUFA High HUFA
• Survival to a SST was higher and gill area was larger in PL fed Artemia nauplii enriched with medium HUFA levels.
• The medium HUFA diet had higher DHA/EPA ratio (1.4 vs. less than 1)
40
60
80
100
120
140
160
SST survival (%) Gill area (µm2/mm)
a a
b
a ab
Could DHA be more important than total HUFA for osmoregulation?
Palacios et al. 2004b
• Na+/K+-ATPase pump activity was higher in PL exposed to low salinity and fed HUFA-enriched diets
• The Na+/K+-ATPase pump activity is affected by an interaction between HUFA levels and salinity.
SST and HUFA
0
5
10
15
20
25
30
35
30 psu 5 psu
Na+
/K+-
AT
Pase
(µM
P/m
g pr
otei
n/h)
Low HUFA Medium HUFA High HUFA
a
c bcbc
a
ab
Loeza et al. 200572
76
80
84
88
5 psu 30 psu 50 psu
Wat
er c
onte
nt g
ills (
%)
a a aaab
b
• In a parallel experiment, water content in gills was affected by HUFA-rich diets, with less water loss in PL fed low levels of HUFA and exposed to high salinities.
• This could indicate lower permeability to water in membranes with saturated fatty acids.
Osmoregulation and HUFA• There is a negative correlation between
Na+/K+-ATPase pump activity and DHA concentration in phospholipid membranes of gills (r2=0.87) during a 15 h salinity acclimation, but not after 3 weeks.
• Similarly, an inhibitory activity of HUFA was found for Na+/K+-ATPase in Artemia salina, irrespective of its developmental stages Morohashi et al. 1991
• Why does DHA affect the activity of the Na+/K+-ATPase pump?
• How do fatty acids modulate water (and ion) permeability?
0 5 10 15 20
DHA concentration (µg/mg)
0
5
10
15
20N
a+/K
+-A
TPas
e (µ
MPi
/g.h
)
Shrimp exposed to changes in salinity after a 15 h (circles) or 3 w (triangles) acclimation period.
Hurtado et al. 2004
Feller et al. 2002
HUFA and osmoregulation• DHA has hundreds of high-
probability conformations in a membrane bilayer. Feller et al. 2002
• This may explain why high levels of DHA are associated with increased speed in many processes occurring across the membrane.
• This is characteristic of DHA; the above properties were lost when substituting with EPA Eldho et al. 2003 Turner et al. 2003
Na+/K+-ATPase activity:• Na+/K+-ATPase pump
activity is increased by DHA in vertebrates
Permeability to water or ions:• Positive correlation between
DHA content in membranes and permeability to Na+ Hulbert 2003
Haines 1994; 2001• Saturated fatty acids decrease
water permeability Haines 2001
Increased tolerance to salinity changes
Increased survival to a SST
More developed osmoregulatory mechanisms
Better physiological condition
Quantity Quality
Larger gill area ~Na+/K+-ATPase activity~Water permeability
?
TG reserves HUFA gill membranes
≠
Or in other words….
• Maybe tolerance to salinity and survival to a SST do not depend on the same osmoregulatory mechanisms.
• Can these differences be the result of time-course activation?
• 1 h: Decreased water permeability in gills of crabs Cantelmo 1977
• 3-6 h: Stabilization of osmotic pressure in shrimp PL Charmantier et al. 1988
• 3 h-24h: Increased Na+/K+-ATPase pump activity in gills of crabs Holliday 1985; Lucu & Flik 1999
• 12 h: PS decarboxilation and PE methylation in gills of crabs Zwingelstein et al. 1998
• 24 h: Increased DHA levels in PC of intestine in fish Leray et al. 1984
• 48 h: Ionocytes differentiation in posterior gills of crabs Genovese et al. 2000; Luquet et al. 2002
MINUTES(Acute exposure)
HOURS(Prolonged exposure)
DAYS-WEEKS(Chronic exposure)
Most SST
Acclimation procedures, e.g. freshwater/inland stocking
Adaptation capacity during estuarine invasion and inland growout
• < 10 m: Electrical potential differences in gills of crabs Siebers et al. 1985; Onken 1996; Luquet et al. 2002; Onken & McNamara 2002
• < 30 m: Swelling of crab cells Gilles & Péqueux 1981
• 5-30 m: Oxidation of proteins in shrimp PL Gomez-Jimenez et al. 2004
• 30 m: Na+/K+-ATPase pump modulation by lipids in fish Towle 1977• Membrane potential • Swelling • Oxidation of reserves• ATPase pump modulation
• Water permeability • Osmotic pressure • Phosholipid metabolism• ATPase activity • HUFA levels • Ionocytes differentiation
• 1-2 w: Ionocytes proliferation Copeland & Fitzjarrel 1968
• 2 w: Increased PS and PE levels in posterior gills Chapelle et al. 1976; Péqueux & Chapelle 1982
• 3 w: Increased synthesis Na+/K+-ATPase pump Neufeld et al. 1980; Lucu & Flik1999
• Ionocytes proliferation • PS & PE changes• Synthesis ATPase
When organisms are exposed to decreased salinities….
Conclusions
• SST can be used to predict survival during stocking of PL, but not performance during growout.
• SST is determined by the physiological condition, that does not necessarily involve osmoregulatory mechanisms, e.g. Na+/K+-ATPase pump activation.
• Time-course changes in salinity (30 m, 24 h, or 1 w) activate different osmoregulatory mechanisms that might be differentially affected by an experimental treatment.
Thank you!
• Larvi’05 organization committee
• Funding by CONACYT & IFS
• Ghent University for ICES emulsion donation
• Genetic program of CIBNOR for larvae donation
• CIBNOR supporting personnel
