Salinity stress response in shrimp postlarvae: Relation to further performance and physiological basis

Transcription

1 Salinity stress response in shrimp postlarvae: Relation to further performance and physiological basis 30 Aniversario Elena Palacios and Ilie S. Racotta CIBNOR, Mexico

2 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. Seawater Diluted sea water SST is based on the life cycle Postlarvae of shrimp: At PL stage, shrimp invade estuaries, where they are exposed to abrupt changes in salinity. 1 L containers PL/replica Recount of live PL

3 SST and further performance Higher survival to SST is associated to greater survival during stocking a and better performance (growth and survival) during growout b, but this has not been tested in controlled conditions a Fegan 1992; b Aquacop et al 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 PL15 a ; PL1 to PL20 b ) a Rees et al. 1994; b Racotta et al This is surprising, given the general belief that SST is predictive of performance to stocking and growout.

4 SST and further performance Our first questions were: Is survival to SST correlated to: survival during stocking? performance during growout? n = 60 Batches (Individual spawns) reared separately from N to PL SST PL15 n = 40 batches (x triplicates n=50 PL) Stocking: in buckets Cumulative Mortalities 10 days, PL were not fed n = 40 batches Growout: in 1m 2 cement tanks (nurseries) 2 months n = 40 batches Alvarez et al. 2004

5 Batches with higher survival to a SST also had higher survival during stocking (r=0.34). 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 Alvarez et al. 2004

6 What does a better physiological condition mean? What are we evaluating when using a SST?

7 The tolerance of PL to changes in salinity is associated to increased osmoregulatory capacities Taken from Bouaricha et al there is a strong correlations between salinity tolerance, the development of osmoregulatory epithelia, the ability to osmoregulate, and activity of Na+/K+- ATPase pump In accordance, when comparing SST in different studies, the older the PL, the higher the survival. For review: Racotta et al salinity tolerance and osmoregulation appear correlated reaching a maximum at PL10-12 Taken from Charmantier et al. 1988

8 Osmoregulatory models for crabs... Morris, 2001 (marine and terrestrial crabs) Towle & Weihrauch, 2001 (crabs) Péqueux 1995 (crabs) When crabs are placed in diluted seawater, there is an influx of water 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.

9 In shrimp PL Na+/K+-ATPase activity (um P/h) A B Alvarez et al psu 5 psu High survival SST Low survival SST 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.

10 In shrimp PL Survival to a SST was correlated to size of PL (r=0.62), even in PL of the same age Survival to a SST was correlated to PL gill area (r=0.55), even when corrected for size Alvarez et al. 2004

11 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?

12 SST and energy reserves Assuming 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? To test this, a SST was applied to 24 h starved PL and to fed PL. Palacios et al. 2004a Fed (3 hrs before sampling) Starved (24 hrs before sampling) n = 6 batches (x triplicates n=50 PL20) SST PL20 Sampling SST PL20 Sampling

13 SST and energy reserves TG (mg/g) a b b b 30 psu 5 psu Fed PL Starved PL 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 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. TG (mg/g) Palacios et al. 2004a a ab b b 30 psu 5 psu Fed PL Starved PL

14 Survival SST (%) b Fed PL SST and energy reserves 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. a Starved PL As expected, survival to a SST was higher in fed PL. More reserves did result in better survival, but survival to a SST cannot be fully explained by decreased Na+/K+- ATPase pump activity Na+/K+-ATPase (µm P/g/h) b Palacios et al. 2004a a 30 psu 5 psu Fed PL c Starved PL c

15 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?

16 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

17 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%) Medium HUFA (30%) SST PL20 Sampling High HUFA (50%) n = 9 batches (x triplicates n=50 PL20) Palacios et al. 2004b

18 HUFA (%) a b b 30 psu 5 psu Low HUFA Medium HUFA High HUFA SST and HUFA a b b 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 DHA (%) A B DHA was the only fatty acid affected by salinity, with lower levels in PL exposed to low salinity 0 30 psu 5 psu Palacios et al. 2004b

19 SST and HUFA Rearing survival (%) Weight (mg) Low HUFA Medium HUFA High HUFA a a b a a SST survival (%) Gill area (µm2/mm) Palacios et al. 2004b b No significant differences were found for rearing performance (survival and growth) between HUFAenriched diets. Could DHA be more important than total HUFA for osmoregulation? 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)

20 SST and HUFA Na+/K+-ATPase (µm P/mg protein/h) Water content gills (%) a a ab bc c bc 30 psu 5 psu Low HUFA Medium HUFA High HUFA a a a a ab b 5 psu 30 psu 50 psu 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. Palacios et al. 2004b 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. Loeza et al. 2005

21 Osmoregulation and HUFA Na+/K+-ATPase (µmpi/g.h) There is a negative correlation between Na+/K+-ATPase pump activity and DHA concentration in phospholipid membranes of gills (r 2 =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 Why does DHA affect the activity of the Na+/K+-ATPase pump? DHA concentration (µg/mg) Shrimp exposed to changes in salinity after a 15 h (circles) or 3 w (triangles) acclimation period. Hurtado et al How do fatty acids modulate water (and ion) permeability?

22 HUFA and osmoregulation Feller et al DHA has hundreds of highprobability conformations in a membrane bilayer. Feller et al 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 Turner et al Permeability to water or ions: Positive correlation between DHA content in membranes and permeability to Na+ Hulbert 2003 Na+/K+-ATPase activity: Na+/K+-ATPase pump activity is increased by DHA in vertebrates Haines 1994; 2001 Saturated fatty acids decrease water permeability Haines 2001

23 Quantity TG reserves Quality HUFA gill membranes Better physiological condition? Larger gill area ~Na+/K+-ATPase activity ~Water permeability More developed osmoregulatory mechanisms Increased tolerance to salinity changes Increased survival to a SST

24 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 timecourse activation?

25 When organisms are exposed to decreased salinities. MINUTES (Acute exposure) Membrane potential Swelling Oxidation of reserves ATPase pump modulation Most SST DAYS-WEEKS (Chronic exposure) 1-2 w: Ionocytes proliferation Copeland & Fitzjarrel w: Increased PS and PE levels in posterior gills Chapelle et al. 1976; Péqueux & Chapelle w: Increased synthesis Na+/K+- HOURS (Prolonged exposure) ATPase pump Neufeld et al. 1980; Lucu & Flik 1 h: Decreased 1999 water permeability in gills of < 10 m: Electrical potential differences in gills of crabs Siebers et al. 1985; Onken crabs 1996; Cantelmo Luquet 1977 et al. 2002; Onken & McNamara 2002 < 30 m: Swelling 3-6 h: of Stabilization crab cells of osmotic Ionocytes pressure proliferation Water permeability Gilles & Péqueux 1981 in shrimp 5-30 m: Oxidation PL Osmotic Charmantier of pressure proteins et al in shrimp PL PS & PE changes Gomez-Jimenez et al m: Na+/K+-ATPase 3 Phosholipid h-24h: Increased pump metabolism modulation Na+/K+-ATPase by Synthesis lipids in ATPase fish pump Towle 1977 ATPase activity activity in gills of crabs Holliday 1985; Lucu & Flik 1999 HUFA levels 12 Ionocytes h: PS differentiation decarboxilation and PE methylation in gills of crabs Zwingelstein et al h: Increased DHA levels in PC of intestine in Adaptation capacity fish Leray et al during estuarine invasion Acclimation procedures, 48 h: Ionocytes differentiation in posterior gills of and inland growout e.g. freshwater/inland crabs Genovese et al. 2000; Luquet et al stocking

26 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.

27 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